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Enlightening the black and white: species delimitation and UNITE species hypothesis testing in the Russula albonigra species complex

December 2021
IMA Fungus 12(1)

DOI:10.1186/s43008-021-00064-0

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CC BY 4.0

Authors:

Ruben De Lange

State Museum of Natural History Stuttgart

Slavomir Adamcik

Slovak Academy of Sciences

Katarína Adamčíková

Slovak Academy of Sciences

Pieter Asselman

Ghent University

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Russula albonigra is considered a well-known species, morphologically delimited by the context of the basidiomata blackening without intermediate reddening, and the menthol-cooling taste of the lamellae. It is supposed to have a broad ecological range and a large distribution area. A thorough molecular analysis based on four nuclear markers (ITS, LSU, RPB2 and TEF1-α) shows this traditional concept of R . albonigra s. lat. represents a species complex consisting of at least five European, three North American, and one Chinese species. Morphological study shows traditional characters used to delimit R . albonigra are not always reliable. Therefore, a new delimitation of the R . albonigra complex is proposed and a key to the described European species of R . subgen. Compactae is presented. A lectotype and an epitype are designated for R . albonigra and three new European species are described: R . ambusta , R . nigrifacta, and R . ustulata . Different thresholds of UNITE species hypotheses were tested against the taxonomic data. The distance threshold of 0.5% gives a perfect match to the phylogenetically defined species within the R. albonigra complex. Publicly available sequence data can contribute to species delimitation and increase our knowledge on ecology and distribution, but the pitfalls are short and low quality sequences.

Maximum Likelihood (ML) tree of Russula sect. Nigricantinae, based on ITS sequence data. ML bootstrap values > 60 are shown. Colour bars of similar colour within each column represent individual UNITE species hypotheses. Missing bars represent unresolved clustering. Samples in colour are representative sequences labelled with SH numbers. Grey shaded samples in white are plotted to the tree based on similarities in distinguishing nucleotide positions (uncertain positions are labelled by grey dotted lines)

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Basidiomata. a-b Russula nigrifacta (RDL 16-044, holotype). c-f Russula albonigra (c: SAV F-20197, epitype; d PRM 934322; e PRM 924409; f SAV F-20177). g Russula ambusta (FH 2008 ST01). h Russula ustulata (AV 16-019, holotype)

…

Russula albonigra (SAV F-755, SAV F-2559, SAV F-20177), hymenium. a Basidia. b Marginal cells. c Basidiospores. d Cystidia near lamellae edges. e Cystidia on lamellae sides. Bar = 10 μm, except for c 5 μm

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Russula albonigra (SAF F-755, SAV F-20177), hyphal terminations of the pileipellis. a Near the pileus margin. b Near the pileus centre. Bar = 10 μm

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Russula albonigra (SAV F-20177), pileocystidia. a Near the pileus margin. b Near the pileus centre. Bar = 10 μm

…

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R E S E A R C H Open Access

Enlightening the black and white: species

delimitation and UNITE species hypothesis

testing in the Russula albonigra species

complex

Ruben De Lange

, Slavomír Adamčík

, Katarína Adamčíkova

, Pieter Asselman

, Jan Borovička

4,5

Lynn Delgat

1,6

, Felix Hampe

and Annemieke Verbeken

ABSTRACT

Russula albonigra is considered a well-known species, morphologically delimited by the context of the basidiomata

blackening without intermediate reddening, and the menthol-cooling taste of the lamellae. It is supposed to have a broad

ecological range and a large distribution area. A thorough molecular analysis based on four nuclear markers (ITS, LSU, RPB2

and TEF1-α) shows this traditional concept of R.albonigra s. lat. represents a species complex consisting of at least five

European, three North American, and one Chinese species. Morphological study shows traditional characters used to delimit

R.albonigra are not always reliable. Therefore, a new delimitation of the R.albonigra complex is proposed and a key to the

described European species of R.subgen.Compactae is presented. A lectotype and an epitype are designated for R.

albonigra and three new European species are described: R.ambusta,R.nigrifacta, and R.ustulata. Different thresholds of

UNITE species hypotheses were tested against the taxonomic data. The distance threshold of 0.5% gives a perfect match to

the phylogenetically defined species within the R. albonigra complex. Publicly available sequence data can contribute to

species delimitation and increase our knowledge on ecology and distribution, but the pitfalls are short and low quality

sequences.

KEYWORDS: Basidiomycota, Coalescent species delimitation, Ectomycorrhizal fungi, New species, Phylogeny, Russulaceae,

Russulales,Russula subgen. Compactae, Integrative taxonomy, Typification, New taxa

INTRODUCTION

Molecular identification of species gained importance over

the last decades (Matute and Sepulveda 2019). As new tech-

niques became available and more easily accessible, the num-

ber of publications using sequence data increased immensely

(Hibbett et al. 2011; Kõljalg et al. 2013; Nilsson et al. 2018).

The main problems with molecular identification are poor

taxon coverage and misidentifications in many public

sequence databases, as well as high infraspecific variability of

DNA regions causing poor performance of the barcoding

gap (Kõljalg et al. 2005; Badotti et al. 2017;Hofstetteretal.

2019). To overcome some of these problems, the UNITE

database was created (https://unite.ut.ee/). UNITE targets the

still most widely used, universal fungal barcode: the nuclear

internal transcribed spacer (ITS) region to provide high-

quality reference records (Nilsson et al. 2018). UNITE groups

individual ITS sequences into species hypotheses (SHs) at

several distance thresholds (i.e. between 0 and 3%), each

assigned a unique digital object identifier (DOI) which allows

unambiguous reference across studies. These species hypoth-

eses are either assigned automatically with a representative

sequence or manually by a taxonomic expert with a

reference sequence (Kõljalg et al. 2013; Nilsson et al. 2018).

which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give

appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if

changes were made. The images or other third party material in this article are included in the article's Creative Commons

licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons

licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain

permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

* Correspondence: Ruben.DeLange@UGent.be

Research Group Mycology, Department of Biology, Ghent University, K.L.

Ledeganckstraat 35, 9000 Ghent, Belgium

Full list of author information is available at the end of the article

Fun

De Lange et al. IMA Fungus (2021) 12:20

https://doi.org/10.1186/s43008-021-00064-0

Most Basidiomycota, the second largest phylum of

fungi, are Agaricomycotina (Naranjo-Ortiz and Gabaldon

2019). A considerable part of this subphylum’s diversity

is concentrated in large genera of ectomycorrhiza-

forming agarics, e.g. Cortinarius and Russula, which ex-

hibit very different evolutionary rates (Ryberg and

Matheny 2012; Varga et al. 2019). Recent studies on lin-

eages of closely related Russula members revealed that

they often comprise closely related species diversified by

ecological adaptation and isolation by distance or dis-

junction (Adamčík et al. 2016b; Caboňet al. 2019;

Looney et al. 2020). This makes the recognising of these

species relevant, despite their similarity in the ITS bar-

code. The efforts to barcode fungal species sometimes

fail, due to lack of taxonomic studies addressing the spe-

cies concept based on type collections (Kõljalg et al.

2020). Our current studies on Russula subgen. Compac-

tae recognised Russula albonigra as one such example.

Its morphological concept is historically established, and

the species is traditionally recognised by the moderately

distant, relatively narrow lamellae, the menthol-cooling

taste of the lamellae, and the surface of the cap and the

stipe, as well as the lamellae, that are strongly and rap-

idly blackening (hence, resulting in black-and-white con-

trast of bruised and untouched parts). Most publications

report blackening without intermediate reddening of the

context and surface (Romagnesi 1967; Adamčík and

Buyck 2014). Microscopically, the species is defined by a

low reticulate spore ornamentation and pileocystidia

without apical knobs (Romagnesi 1967). However, our

current search in two databases, which seek to adopt a

nomenclatural concept for fungal operational units de-

fined by the ITS barcode, recovered inconsistencies.

Samples identified as R. albonigra in the BOLD database

(https://www.boldsystems.org/) do not match any of the

species hypothesis under this species name published in

UNITE (https://unite.ut.ee/), and vice versa.

In this study, we use sequence data of four DNA

markers and a detailed morphological revision to test

the taxonomic status within the R. albonigra complex.

To test different distance thresholds of UNITE species

hypotheses, we used the strict genealogical concordance

assessing the extent of genetic concordance across loci,

the coalescent based species delimitation modelling the

genealogical history of individuals back to a common

ancestor and morphological differences.

MATERIAL AND METHODS

Sampling

This study is based on collections from sampling trips

in Belgium (2016, 2017 and 2018), Italy (1997, 2000

and 2016), Norway (2016), Slovakia (2003, 2006, 2008,

2009, 2011, 2015 and 2017), and Sweden (2016 and

2018). All collections are deposited in the Herbarium

Universitatis Gandavensis (GENT) or the Slovak

Academy of Sciences (SAV). Supplementary collec-

tions were requested from the Mycological Depart-

ment of the National Museum in Prague, Czech

Republic (PRM), and from the personal collections of

Felix Hampe, Jesko Kleine, and Helga Marxmüller

(the latter recently deposited in the State Museum of

Natural History Karlsruhe, KR).

Samples that could belong in the Russula albonigra

complex were selected based on morphology: (1) col-

lections that were identified as R.albonigra in the

field based on macro-morphology; and (2) fungarium

collections labelled as R.albonigra based on both

macro- and micro-morphological observations. The

selected samples were molecularly checked using the

ITS marker as a guideline and not included for

further study when not placed within the R.albonigra

complex.

Morphological analysis

The macroscopic description is based on observations

from fresh material, with colour codes following

Kornerup and Wanscher (1978), guaiac reactions refer-

ring to Chalange (2014), and spore print colour codes

following the scale of Romagnesi (1967). The micro-

scopic description and terminology follow Adamčík

et al. (2019). Microscopic characters were studied from

dried material, spores were observed in Melzer’s reagent,

elements of the hymenium and pileipellis were observed

in Congo red in L4 after ca. 10 s in KOH 10%. Basidio-

spores were measured using a crosshair eyepiece on a

Zeiss Axioskop 2 microscope. Line drawings of spores

were made based on stacked photographs (Nikon Eclipse

Ni-U microscope, stacking software: Extended Depth of

Field, Nikon Nis Elements module) at an original magni-

fication of 5000×. Measurements of other elements were

made using an eyepiece micrometer and line drawings

were prepared with the aid of a camera lucida (Olympus

U-DA) on an Olympus CX21 or BX43 microscope, at

original magnifications of 1500× or 2000×. Tissues were

mounted in Cresyl Blue (Buyck 1989), sulfovanillin

(Cabon et al. 2017) and treated with carbolfuchsin

(Romagnesi 1967) to observe the presence and colour

changes of incrustations and cystidium contents. All

cited collections in the species descriptions have been

sequenced, at least for ITS.

The key provided in the Taxonomy section is based on

our observations of species within the R.albonigra com-

plex, and following the traditional species concepts in

literature for the other taxa. Russula clementinae is not

included in the key as it is interpreted as a synonym of

R.densifolia (Sarnari 1998).

De Lange et al. IMA Fungus (2021) 12:20 Page 2 of 31

Molecular analysis

DNA extraction and amplification was performed in

either the molecular laboratory of Ghent University or

that of the Slovak Academy of Sciences. Sequencing of

PRM collections was conducted within the study of

Leonhardt et al. (2019).

In Ghent University, DNA from fresh material was ex-

tracted using the CTAB extraction described in Nuytinck

and Verbeken (2003). DNA from dried material was ex-

tracted using a modified CTAB protocol (Tel-Zur et al.

1999; modified by Meise Botanic Garden and Research

Group Mycology of Ghent University). The original proto-

col was optimized for cacti species. These plant types tend

to have larger cells compared to fungi. Hence, the ratio

DNA content:biomass for fungi is much higher. There-

fore, less biomass is needed as starting material and buffer

volumes were adjusted consequently. Because mucilagi-

nous polysaccharides have not been observed in previous

Fungal DNA extractions the use of sorbitol in our extrac-

tions was omitted. Additionally, the use of CTAB as deter-

gent to brake open fungal cells seemed to suffice to access

the fungal DNA in an efficient way. Hence, the extra sar-

kosyl added in the lysis step was also omitted from our

protocol. Protocols for PCR amplification follow Le et al.

(2007). In the Slovak Academy of Sciences, total genomic

DNA was extracted from dried material using the EZNA

Fungal DNA Mini Kit (Omega Bio-Tek, Norcross, GA,

USA) following the manufacturer’s instruction. Amplifica-

tion of DNA was performed in a PCR reaction mix con-

sisting of approximately 2 ng/μloftemplateDNA,forward

and reverse primers (10 pmol/μl), 5× HOT FIREPol® Blend

Master Mix (Solis BioDyne, Tartu, Estonia) and molecular

grade water added up to 20 μl. Four nuclear markers were

amplified: (1) the internal transcribed spacer region of

ribosomal DNA (ITS), comprising the ITS1 and ITS2 spa-

cer regions and the ribosomal gene 5.8S, using primers

ITS1-F and ITS4 (White et al. 1990; Gardes and Bruns

1993); (2) a part of the ribosomal large subunit 28S region

(LSU), using primers LR0R and LR5 (Moncalvo et al.

2000); (3) the region between the conserved domains 6

and 7 of the second largest subunit of the RNA polymer-

ase II (RPB2), using primers bRPB2-6F and fRPB2–7cR or

bRPB2–7.1R (Liu et al. 1999;Matheny2005); and (4) the

translation elongation factor 1-alpha (TEF1α), using pri-

mer pairs EF1-1018F and EF1-1620R or tef1F and tef1R

(Morehouse et al. 2003; Stielow et al. 2015). PCR products

from Ghent University were sequenced using an auto-

mated ABI 3730 XL capillary sequencer at Macrogen. In

the Slovak Academy of Sciences, the PCR products were

purified using Qiaquick PCR Purification Kit (Qiagen,

Hilden, Germany) and samples were sequenced by the

Seqme company (Dobříš, Czech Republic).

Forward and reverse sequences were assembled into

contigs and edited where needed with BioloMICS

(BioAware SA NV). All sequences generated were de-

posited in GenBank (Table 1).

Phylogenetic analysis

Identifications of publicly available sequences of fungi

often match contrasting taxonomic species concepts. To

provide reliable sampling in accordance with traditional

species concepts we selected three representative collec-

tions of each European species described within R. sub-

gen. Compactae, identified using the most recent and

reliable keys (Romagnesi 1967; Sarnari 1998). These col-

lections, together with the collections of R. albonigra s.

lat. were sequenced by us (Table 1). For non-European

species, sequences used in Adamčík et al. (2019)or

Buyck et al. (2020) were included if multiple of the

markers used in this study were available for these sam-

ples, with an ITS sequence obligatory. Four species of

Russula subgen. Archaeae were used as an outgroup, be-

cause the recent phylogenies of the genus place this sub-

genus as sister to R. subgen. Compactae.

Sequences were aligned using the online version of the

multiple sequence alignment program MAFFT v7

(Katoh and Toh 2008), using the E-INS-i strategy. Trail-

ing ends of the alignments were trimmed and the align-

ments were manually edited when necessary in MEGA7

(Kumar et al. 2016). The alignments can be obtained

from the first author and TreeBASE (Submission ID

26815). The alignments were partitioned into following

partitions: ITS-LSU-alignment: partial 18S, ITS1, 5.8S,

ITS2, LSU; RPB2-alignment: the RPB2 intron and the

first, second and third codon positions of the exon;

TEF1α-alignment: the first and second intron and the

first, second and third codon positions. PartitionFinder2

was used to find the appropriate partitioning scheme

and substitution models using the Akaike information

criterion (AICc) with a greedy search over all models

(Guindon et al. 2010; Lanfear et al. 2012; Lanfear et al.

2017). Maximum likelihood (ML) analyses were con-

ducted with IQ-Tree (Nguyen et al. 2014; Chernomor

et al. 2016) using standard bootstrapping analysis (1000

replicates). Bayesian inference (BI) was executed with

MrBayes v3.2.6 (Huelsenbeck and Ronquist 2001;

Ronquist and Huelsenbeck 2003). Two independent par-

allel runs of one cold and three heated chains were run

for ten million (single-locus datasets) or twenty million

generations (multi-locus dataset) with a sample fre-

quency of 100. Potential Scale Reduction Factor (PSRF)

values approached 1.0. Convergence and Effective Sam-

ple Size (ESS) statistics of the runs were also examined

with Tracer v1.7.1 (Rambaut et al. 2018). A burn-in sam-

ple of 20% was excluded before constructing the major-

ity rule consensus tree. Analyses were first performed on

each alignment separately and visually checked for in-

congruence. Significant incongruence was assumed if

De Lange et al. IMA Fungus (2021) 12:20 Page 3 of 31

Table 1 Specimens and GenBank accession numbers of DNA sequences used in the multi-locus phylogenetic analysis

Taxon Voucher collection (herbarium) Country ITS LSU RPB2 TEF1α

Russula acrifolia LD 16–022 (GENT) Sweden MW172319 MW182479 MW306683 MW273325

Russula acrifolia RDL 18–012 (GENT) Sweden MW172320 MW182480 MW306684 MW273326

Russula acrifolia RDL 18–021 (GENT) Sweden MW172321 MW182481 MW273327

Russula adusta LD 16–025 (GENT) Sweden MW172316 MW306682 MW273322

Russula adusta RDL 18–020 (GENT) Sweden MW172317 MW182477 MW273323

Russula adusta RDL 18–028 (GENT) Sweden MW172318 MW182478 MW273324

Russula albonigra JK RUS 13090603 (Jesko Kleine*) Germany MW172296 MW182461 MW306670

Russula albonigra SAV F-755 (SAV) Slovakia MW172291

Russula albonigra SAV F-2559 (SAV) Slovakia MW172292

Russula albonigra SAV F-20177 (SAV) Slovakia MW172298 MW182463 MW306672 MW273311

Russula albonigra SAV F-20197 (SAV) Slovakia MW172299 MW182464 MW306673 MW273312

Russula albonigra SAV F-3465 (SAV) Slovakia MW172293 MW182460 MW306669 MW273309

Russula albonigra SAV F-4776 (SAV) Slovakia MW172297 MW182462 MW306671 MW273310

Russula albonigra PRM 934322 (PRM) Czech Republic MW172294

Russula albonigra PRM 924409 (PRM) Czech Republic MW172295

Russula ambusta FH 2008 ST01 (Felix Hampe*) Germany MW172300 MW182465

Russula ambusta SAV F-3358 (SAV) Slovakia MW172301 MW182466

Russula cf. anthracina RDL 16–031 (GENT) Italy MW172313 MW182474 MW306679 MW273319

Russula cf. anthracina RDL 16–058 (GENT) Italy MW172314 MW182475 MW306680 MW273320

Russula cf. anthracina RDL 18–026 (GENT) Sweden MW172315 MW182476 MW306681 MW273321

Russula archaeosuberis BB 12.085 (PC) Italy KY800355° KU237593° KU237878° KU238019°

Russula atramentosa FH 2011-002R (Felix Hampe*) Belgium MW172322 MW182482 MW306685 MW273328

Russula atramentosa RDL 16–050 (GENT) Italy MW172323 MW306686

Russula atramentosa FH0170824–02 (Felix Hampe*) Germany MW172324 MW182483 MW306687 MW273329

Russula camarophylla MPG11–7-09 (PC) Spain KY800353° KU237579° KU237865° KU238006°

Russula cantharellicola UC1999420 United States KF306036°

Russula cascadensis OSC 1064009 (OSC) United States EU526006°

Russula cortinarioides BB 07.133 (PC) United States KP033485° KP033507°

Russula cortinarioides BB 07.103 (PC) United States KP033480° KP033491° KP033502° KU237985°

Russula densifolia RDL 16–001/2 (GENT) Belgium MW172325 MW182484 MW306688 MW273330

Russula densifolia RDL 18–052 (GENT) Belgium MW172326 MW182485 MW306689 MW273331

Russula densifolia RDL 17–024 (GENT) Belgium MW172327 MW182486 MW306690 MW273332

Russula densissima FH 2014 ST04 (Felix Hampe*) Germany MW172328 MW306691

Russula densissima FH 2010 ST02 (Felix Hampe*) Germany MW172329 MW306692

Russula dissimulans BPL704 (TENN) United States KY848513°

Russula earlei BPL245 (TENN) United States KT933961° KT933820° KT933891°

Russula cf. eccentrica BB 07.044 (PC) United States KP033479° KP033490° KP033501° KU237937°

Russula cf. eccentrica BB 07.132 (PC) United States KP033478° KP033489° KP033500° KU237926°

Russula cf. fuliginosa FH RUS 14091001 (Felix Hampe*) Slovakia MW172330 MW182487 MW306693 MW273333

Russula cf. fuliginosa FH RUS 14091201 (Felix Hampe*) Slovakia MW172331 MW182488 MW306694 MW273334

Russula gossypina BB 06.002 (PC) Madagascar KY800350° KU237450° KU237736° KU237886°

Russula ingwa MEL2101936 Australia EU019919°

Russula khanchanjungae AV KD KVP 09–106 (GENT) India KR364129° JN389004° JN375607°

Russula lateriticola BB 06.031 (PC) Madagascar KP033476° KP033487° KP033498° KU237888°

De Lange et al. IMA Fungus (2021) 12:20 Page 4 of 31

two different relationships (one monophyletic and the

other non-monophyletic) for any set of taxa were sup-

ported with bootstrap values (BS) ≥70 or posterior prob-

abilities (PP) ≥90. The resulting gene trees did not show

any supported conflicts, therefore all alignments could

be concatenated. The concatenated alignment was used

for the multi-locus phylogenetic analyses (Fig. 1).

Coalescent species delimitation approaches

For species delimitation under the multispecies coales-

cent model a part of the alignment used in the multi-

locus phylogenetic analyses mentioned above, compris-

ing members of the Russula albonigra complex, was

used. A total of five potential species units (as proposed

by the ML and BI trees) were evaluated as the full

model. Two coalescent species delimitation methods

were performed to test these species hypotheses. The

first method was implemented in Bayesian Phylogenetics

and Phylogeography, BP&P v4.3.8 (Yang 2015). We per-

formed analysis A11 (Yang and Rannala 2014) for un-

guided species delimitation using rjMCMC algorithm 0

(Yang and Rannala 2010). Analyses were run with sev-

eral values for the fine-tune parameter (ε= 2, 5, 10 and

20) and we assigned equal probabilities to the rooted

species trees as a species model prior. Because the prior

distributions on the ancestral population size and root

age can affect the posterior probabilities of the model,

we considered three different combinations of priors

(based on the idea of Leache and Fujita (2010)): (1) θ~

IG(3,0.002) and τ~ IG(3,0.002), (2) θ~ IG(3,0.02) and

τ~ IG(3,0.02), and (3) θ~ IG(3,0.02) and τ~ IG(3,0.002)

(with α= 3 for a diffuse prior as proposed in the man-

ual). For each combination of settings the analysis was

run twice with a different seed (to confirm consistency

between runs) for 200,000 generations (sampling interval

of two) and a burn-in of 50,000. As a second species de-

limitation method we used the STACEY v1.2.5 (Jones

2017) package implemented in BEAST2 (Bouckaert et al.

2019). The xml-file for the BEAST2 runs were prepared

in BEAUTi v2.6.3 (Drummond et al. 2012). We used fol-

lowing partitions: for the nrDNA (1) 5.8S, (2) ITS1 +

ITS2 and (3) LSU; for the protein coding loci the introns

and the first, second and third codon positions of the

exons. PartitionFinder2 was used to find the appropriate

substitution models. The substitution rate of each parti-

tion was estimated independently of the others. Clock

and tree model parameters were estimated independ-

ently for the nrDNA and each protein coding locus. We

used a lognormal, relaxed clock model and a Yule tree

model. The Collapse Height parameter εwas set to

−5

. The Collapse Weight parameter ωwas estimated

and given a uniform prior on [0,1] so that every number

of species between five and one is regarded as equally

likely a priori. We ran five parallel MCMC runs for one

Table 1 Specimens and GenBank accession numbers of DNA sequences used in the multi-locus phylogenetic analysis (Continued)

Taxon Voucher collection (herbarium) Country ITS LSU RPB2 TEF1α

Russula nigricans RDL 17–004 (GENT) Belgium MW172332 MW182489 MW306695 MW273335

Russula nigricans RDL 17–007 (GENT) Belgium MW172334 MW182491 MW306697 MW273337

Russula cf. nigricans RDL 17–005 (GENT) Belgium MW172333 MW182490 MW306696 MW273336

Russula nigrifacta RDL 16–028 (GENT) Italy MW172307 MW306676 MW273316

Russula nigrifacta RDL 16–044 (GENT) Italy MW172308 MW182470 MW306677 MW273317

Russula nigrifacta RDL 16–063 (GENT) Italy MW172306 MW273315

Russula nigrifacta SAV F-2418 (SAV) Slovakia MW172304

Russula nigrifacta SAV F-2419 (SAV) Slovakia MW172303 MW182468

Russula nigrifacta SAV F-1501 (SAV) Slovakia MW172302 MW182467 MW306674 MW273314

Russula nigrifacta SAV F-3006 (SAV) Slovakia MW172305 MW182469 MW306675

Russula polyphylla BB 07.134 (PC) United States KP033486° KP033497° KP033508° KU238023°

Russula polyphylla BB 07.023 (PC) United States KP033481° KP033492° KP033503° KU237986°

Russula roseonigra KR-M-0042973/MxM R-9308 (KR) France MW172335

Russula roseonigra FH 2014 ST01 (Felix Hampe*) Germany MW172336 MW306698 MW273338

Russula roseonigra RDL 16–024 (GENT) Italy MW172337 MW182492 MW306699 MW273339

Russula sp. FH 12–064 (GENT) Thailand MN130076° MN380517°

Russula sp. 1 RW 1975 (GENT) Italy MW172309 MW182471

Russula ustulata AV 16–019 (GENT) Norway MW172312 MW182473 MW306678 MW273318

Russula ustulata SAV F-2610 (SAV) Italy MW172310 MW182472

Russula ustulata PRM 924452 (PRM) Czech Republic MW172311

In bold: types; * personal herbarium; ° sequences not generated in this study

De Lange et al. IMA Fungus (2021) 12:20 Page 5 of 31

Fig. 1 Maximum Likelihood (ML) tree of Russula subgen. Compactae, based on concatenated ITS, LSU, RPB2 and TEF1αsequence data. ML bootstrap values > 75 and BI posterior probabilities > 0.95

are shown

De Lange et al. IMA Fungus (2021) 12:20 Page 6 of 31

0.03

KX441086 China

UDB0641772 Estonia

JF519253 Austria

UDB038057 NOBAS948-15 O-F-249610 Norway

KF306042 United States

JF908707 Italy

KF306043 United States

JF519235 Austria

JF519171 Austria

UDB0367026 Estonia

JF519248 Austria

UDB0392273 Estonia

KT800130 United States

UDB0448007 Estonia

DQ422029 Sweden

UDB0669827 Estonia

HM488592 United States

KJ769287 Russian Federation

UDB031523 Estonia

UDB0513905 Estonia

UDB011193 Estonia

NOBAS1861-16 O-F-251464 Norway

UDB051783 Estonia

UDB0287633 Estonia

JF519228 Austria

KF306041 United States

KF306040 United States

NOBAS1778-16 O-F-251577 Norway

UDB051911 Estonia

UDB063604 Estonia

NOBAS1869-16 O-F-251473 Norway

JF834364 United States

UDB0661958 Estonia

JF834355 United States

UDB0211768 Estonia

UDB0703616 Estonia

UDB031522 Estonia

UDB0660450 Estonia

NOBAS4607-17 O-F-253969 Norway

UDB0299885 Estonia

UDB0701696 Estonia

UDB0585705 Estonia

UDB0459908 Estonia

FJ013070 Spain

UDB0536051 Estonia

UDB016040 Finland

AB291763 Japan

UDB037058 NOBAS3604-16 O-F-26206 Norway

UDB0602964 Estonia

UDB024525 Laos

UDB011240 Estonia

UDB0273348 Estonia

UDB0703036 Estonia

100

99 100

100

0.5% SH1961382.08FU

<0.5% SH2310095.08FU

1% SH1740694.08FU

1.5% SH1569600.08FU

2% SH1425818.08FU

2.5% SH1300493.08FU

3% SH1188805.08FU

<0.5% SH2310133.08FU

0.5% SH1961409.08FU

<0.5% SH2310129.08FU

<0.5% SH2310126.08FU

0.5% SH1961425.08FU

0.5% SH1961426.08FU

<0.5% SH2310140.08FU

0.5% SH2890350.08FU

1% SH1740706.08FU

1.5% SH1569600.08FU

2% SH1425829.08FU

2.5% SH1300506.08FU

3% SH1188820.08FU

0.5% SH1961392.08FU

<0.5% SH3011319.08FU

<0.5% SH2310110.08FU

MH930188 Russian Federation

UDB065518 Estonia

UDB0557800 Estonia

UDB0502905 Estonia

UDB0663165 Estonia

representative sequence locked

R. albonigra R. ustulata R. ambusta R. nigrifacta

<.5

0.5

1.0

1.5

2.5

2.0

3.0

R. albonigra lineage

0.5% SH1961425.08FU

Russula nigrifacta SAV F-3006 Slovakia

Russula nigrifacta SAV F-1501 Slovakia

Russula nigrifacta RDL 16-028 Italy

Russula nigrifacta RDL 16-044 Italy HOLOTYPE

Russula nigrifacta SAV F-2418 Slovakia

Russula nigrifacta SAV F-2419 Slovakia

Russula nigrifacta RDL 16-063 Italy

Russula ambusta FH 2008 ST01 Germany

Russula ambusta SAV F-3358 Slovakia HOLOTYPE

Russula ustulata AV 16-019 Norway HOLOTYPE

Russula ustulata PRM 924452 Czech Republic

Russula ustulata SAV F-2610 Italy

Russula albonigra SAV F-20177 Slovakia

Russula albonigra JK RUS 13090603 Germany

Russula albonigra SAV F-3465 Slovakia

Russula albonigra SAV F-4776 Slovakia

Russula albonigra SAV F-755 Slovakia

Russula albonigra PRM 934322 Czech Republic

Russula albonigra SAV F-20197 Slovakia EPITYPE

Russula albonigra SAV F-2559 Slovakia

Russula albonigra PRM 924409 Czech Republic

Russula sp. 1 RW 1975 Italy

Russula roseonigra KR-M-00242973/MxM R-9308 France

Russula roseonigra RDL 16-024 Italy

Russula roseonigra FH 2014 ST01 Germany

Russula cf. anthracina RDL 16-031 Italy

Russula cf. anthracina RDL 18-026 Sweden

Russula cf. anthracina RDL 16-058 Italy

Russula acrifolia LD 16-022 Sweden

Russula acrifolia RDL 18-012 Sweden

Russula acrifolia RDL 18-021 Sweden

Russula cascadensis EU526006 United States

Russula adusta LD 16-025 Sweden

Russula adusta RDL 18-028 Sweden

Russula adusta RDL 18-020 Sweden Russulaaff. densifolia

Russula aff. densifolia AB291759 Japan

Russula aff. densifolia AB291758 Japan

AB291755 JapanRussula aff. densifolia

Russula sp. FH 12-064 Thailand

Russula atramentosa FH 2011-002R Belgium

Russula atramentosa RDL 16-050 Italy

Russula atramentosa FH0170824-02 Germany

Russula aff. densifolia AB291762 Japan

Russula aff. densifolia AB291756 Japan

Russula densissima FH 2014 ST04 Germany

Russula densissima FH 2010 ST02 Germany

Russula densifolia RDL 16-001/2 Belgium

Russula densifolia RDL 17-024 Belgium

Russula densifolia RDL 18-052 Belgium

Russula aff. densifolia AB291761 Japan

Russula aff. densifolia AB291760 Japan

Russula aff. densifolia AB291757 Japan

Russula cf. fuliginosa FH RUS 14091001 Slovakia

Russula cf. fuliginosa FH RUS 14091201 Slovakia

Russula dissimulans KY848513 United States

Russula nigricans RDL 17-004 Belgium

Russula nigricans RDL 17-007 Belgium

Russula cf. nigricans RDL 17-005 Belgium

Fig. 2 Maximum Likelihood (ML) tree of Russula sect. Nigricantinae, based on ITS sequence data. ML bootstrap values > 60 are shown. Colour bars of

similar colour within each column represent individual UNITE species hypotheses. Missing bars represent unresolved clustering. Samples in colour are

representative sequences labelled with SH numbers. Grey shaded samples in white are plotted to the tree based on similarities in distinguishing

nucleotide positions (uncertain positions are labelled by grey dotted lines)

De Lange et al. IMA Fungus (2021) 12:20 Page 7 of 31

billion generations sampling every 1000th tree. Conver-

gence and Effective Sample Size (ESS) statistics of the

runs were examined with Tracer v1.7.1. Twenty percent

of each run was discarded as burn-in and the remaining

posterior samples were combined using LogCombiner

v2.6.3 (Drummond and Rambaut 2007) and used to cal-

culate the most likely number of clusters (i.e., putative

species), using SpeciesDelimitationAnalyzer (Jones et al.

2014).

Species hypothesis and threshold testing

ITS sequences generated by authors of this study and

used in the multi-locus phylogeny were combined with

all ITS sequences, either labelled as R.albonigra or

showing high similarity (97%) to our sequences of

R. albonigra s.lat., available on UNITE (https://unite.ut.ee/),

GenBank (www.ncbi.nlm.nih.gov)andBOLD(https://www.

boldsystems.org/) databases. Accession numbers are given

in Fig. 2. The clade containing R. nigricans and R. dissimu-

lans is in a basal position within R.sect.Nigricantinae (see

Fig. 1) and is chosen as the outgroup. Short sequences

(containing only ITS1 or ITS2) and sample UDB065518

(containing many ambiguities and differences in conserved

domains compared to other sequences of the group)

were excluded from the analysis. The sequences were

aligned following the same principles as mentioned

above. The alignment was partitioned into following

partitions: ITS1, 5.8S and ITS2. A ML analysis was con-

ducted with IQ-Tree (Nguyen et al. 2014;Chernomor

et al. 2016) using the option to first test for the best

substitution model (Kalyaanamoorthy et al. 2017)and

standard bootstrapping analysis (1000 replicates). The

excluded samples were later plotted on the tree based

on similarities in distinguishing nucleotide positions

(Additional file 1). We identified the single nucleotide

positions distinguishing the species of the R.albonigra

complex and compared the excluded samples to the

good quality sequences based on these positions to esti-

mate their placement in the tree. SH inclusiveness across

sequence distance threshold values, as it is shown on

UNITE, is plotted against the tree.

RESULTS

Multi-locus phylogenetic analyses

All 24 sequences generated by the authors of specimens

putatively identified as R. albonigra, are placed within R.

subgen. Compactae and group together with other mem-

bers of R. sect. Nigricantinae (Fig. 1). All but two of

these sequences are placed in one strongly supported

clade, here further referred to as the R.albonigra com-

plex. The two sequences outside the R.albonigra com-

plex are placed in either the R.atramentosa clade or the

R. anthracina clade (these sequences were not used for

the final analysis and are not shown in the trees). Our

molecular analysis shows the presence of five distinct

European clusters within the R.albonigra complex (Fig.

1). They form four well supported clades and one single-

ton collection on a long branch. The overall topology of

the ML and BI tree was congruent.

The name R.albonigra is assigned to a clade with two

collections from the Czech Republic, PRM 924409

and PRM 934322, that originate from the type collecting

area. Three species, R.ambusta,R.nigrifacta and R.ustulata,

are described here as new. One species is represented by a

singleton position in the tree, placed as sister to R. albonigra

and is labelled as Russula sp. 1. These two species

form a sister clade to a larger clade containing R.ambusta,

R.nigrifacta, and R.ustulata. The relations within the

latter clade are not well supported in the BI tree.

Coalescent-based species delimitation

The full set of proposed species (i.e. five species) was re-

covered as the highest supported species model in the

BP&P analysis under each combination of settings, with

posterior probabilities ranging from 0.91 to 0.99. Also

the STACEY analysis resulted in the highest probability

(posterior probability of 0.99) for five minimal clusters

(species). Both coalescent delimitation methods confirmed

the species hypothesis for all five clusters in the multi-

locus phylogenetic analysis.

ITS analysis and optimal SH distance

When searching for UNITE species hypotheses labelled

as R. albonigra, at every threshold, two species hypoth-

eses are found that are not placed within the R.alboni-

gra complex defined by our multi-locus analysis. Both

are represented by a singleton sequence. The first,

UDB024525 represents a collection from Lao People’s

Democratic Republic and seems more closely related to

R. atramentosa. The second, JF908707 represents a

collection from Italy with an isolated position in the

phylogeny (Fig. 2).

The general topology of the ITS tree is congruent with

the multi-locus tree and all sequences of the R. albonigra

complex generated by the authors of this study are again

placed within this monophyletic group (Fig. 2). The ITS

analyses revealed the presence of three additional North

American clusters and one Chinese collection of singleton

position, within the R. albonigra complex. Two of the

North American clusters are supported and probably rep-

resent well defined species, while the status of the three

sequences from the USA (KF306041, JF834355 and

KF306040) is uncertain and requires more sequence data

to resolve. The singleton Chinese sample (KX441086)

probably represents an undescribed species sister to R.

albonigra. Furthermore, Fig. 2shows an overview of the

different UNITE species hypotheses within the R.alboni-

gra complex at different thresholds. At a threshold of 1%

De Lange et al. IMA Fungus (2021) 12:20 Page 8 of 31

or higher two species hypotheses are recognised (for SH

numbers see red and pink boxes in the Fig. 2). The first

one labelled as R. albonigra with the representative

UNITE sequence UDB016040 covers R.ambusta,R.nigri-

facta,R.ustulata and the North American species. The

second one with the representative sequence JF519228 is

labelled as Russula sp.and covers R.albonigra and the

Chinese species. At a threshold of 0.5%, all European spe-

cies (except for R. sp. 1 which is not represented by any

public sequence) and two North American species are

supported. Threshold < 0.5% gives additional units within

the phylogenetic species that were not supported by our

multi-locus analysis.

TAXONOMY

The species within the Russula albonigra complex are

characterised by the moderately distant, relatively nar-

row lamellae, the context that is rapidly and strongly

blackening, generally without intermediate reddening. In

some cases though, some slight reddening is observed.

The taste of the lamellae and flesh is never acrid, but

can be menthol-cooling. Microscopically, the species are

Fig. 3 Basidiomata. a-b Russula nigrifacta (RDL 16–044, holotype). c-f Russula albonigra (c: SAV F-20197, epitype; dPRM 934322; ePRM

924409; fSAV F-20177). gRussula ambusta (FH 2008 ST01). hRussula ustulata (AV 16–019, holotype)

De Lange et al. IMA Fungus (2021) 12:20 Page 9 of 31

defined by spores with low and dense warts forming

subreticulate to reticulate ornamentation, long pileocys-

tidia (if present) and a cystidial content which is not

reacting in sulfovanillin.

Russula albonigra (Krombh.) Fr., Hymenomyc.eur.

(Upsaliae): 440 (1874). (Figs. 3c-f, 4,5and 6).

Basionym:Agaricus alboniger Krombh., Naturgetr.

Abbild. Beschr. Schwämme (Prague) 9: 27 (1845).

Type: Krombholz (1845)lectotype designated here,

MB10000343); Slovakia: Oblík Nature reserve, with

Fagus, 23 Sept. 2017, S. Adamčík (SAV F-20179 –epi-

type designated here, MB10000342).

Description:Pileus large, 56–112 mm diam., plano-

convex, at the centre with shallow but wide depression;

margin deflexed, long involuted, not striated, smooth; pi-

leus surface velvety and smooth near margin, towards

the centre radially wrinkled or rugulose, centre smooth,

dry, matt, almost not peeling (max. to 1/3 of the radius);

young completely white, later becoming yellowish white

(4A2), cream (4A3) to orange-grey (5B2) at the centre,

more greying and blackening when old. Lamellae seg-

mentiform to subventricose, to 6 mm deep, adnate to

subdecurrent; snow white, later yellowish white (4A2),

blackening with age or when bruised; with numerous

lamellulae of different lengths, frequently forked near

Fig. 4 Russula albonigra (SAV F-755, SAV F-2559, SAV F-20177), hymenium. aBasidia. bMarginal cells. cBasidiospores. dCystidia near lamellae

edges. eCystidia on lamellae sides. Bar = 10 μm, except for c 5 μm

De Lange et al. IMA Fungus (2021) 12:20 Page 10 of 31

the stipe but also near the pileus margin, often anas-

tomosed; moderately crowded to moderately distant,

L = 190–260, l = 1 (one between each pair of long

lamellae); edges even, concolorous, blackening with age.

Stipe 42–60 × 14–29mm, cylindrical, firm and fleshy, lon-

gitudinally striated, velvety near the lamellae; white, later

becoming greyish orange (5B4) near the base; interior

solid, cortex ca. 2.5 mm thick. Context ca. 7 mm thick at

mid-radius, hard, white, turns rapidly grey and then black

on cut section, at surface also turns red before grey and

black; turning orange with FeSO

, immediately dark blue

with guaiac (strong reaction, +++); taste mild, slightly like

mint (refreshing) in lamellae, odour weak of apples. Spore

print white (Ia).

Basidiospores (7.1–)7.5–8.0–8.5(−9.4) × (5.6–)5.9–6.3–6.7(−

7.1) μm, broadly ellipsoid to ellipsoid, Q = (1.16–)1.22–1.29–

1.36(−1.49); ornamentation of low, dense [(6–)7–10(−11) in a

3μm diam circle] amyloid warts, 0.1–0.4 μm high, subreticu-

late, abundantly fused into chains [(0–)3–7(−8) fusions in a

3μm diam circle], and also connected by short, fine line

Fig. 5 Russula albonigra (SAF F-755, SAV F-20177), hyphal terminations of the pileipellis. aNear the pileus margin. bNear the pileus centre. Bar = 10 μm

De Lange et al. IMA Fungus (2021) 12:20 Page 11 of 31

connections [0–4(−6) in a 3 μm diam circle]; suprahilar spot

medium-sized, not amyloid. Basidia (44–)48.9–55.1–

61.3(−75) × 10.0–10.8–11.6(−13) μm, narrowly clavate,

4-spored. Hymenial cystidia (65–)69.6–85.8–

102.0(−115) × (7–)7.6–8.5–9.4(−10) μm, cylindrical to

narrowly fusiform, apically obtuse to mucronate, thin-

walled; with little content composed of large pale, oily, re-

fringent guttules, without reaction in sulfovanillin; near

the lamellae edges, (30–)40.4–58.5–76.6(−98) × (6–)7.1–

7.9–8.7(−9) μm, cylindrical to narrowly fusiform, some-

times slightly flexuose, apically obtuse to mucronate or

with small appendage, thin-walled, content as on lamellae

sides. Lamellae edges sterile, when older elements can

contain brown pigments; marginal cells (11–)16.5–22.1–

27.7(−31) × (3–)3.5–4.9–6.3(−8) μm, poorly differenti-

ated, cylindrical, flexuose, thin-walled. Pileipellis ortho-

chromatic in Cresyl Blue, 80–90 μm deep, not sharply

delimited from trama and not gradually passing, inter-

mediate; subpellis not delimited from suprapellis; hyphae

3–6μm wide near trama, not regular in width, dense,

homogeneous, pigmented only near the surface, with no

distinct gelatinous coating or only weakly on deeper hy-

phae. Acid-resistant incrustations absent. Hyphal termina-

tions near the pileus margin long, with multiple septa,

flexuous, thin-walled, filled with irregular refractive bodies

containing brown pigments; terminal cells very long

(35–)55.9–86.6–117.3(−160) × (5–)5.2–6.6–8.0(−10) μm,

narrowly cylindrical to subulate, on average apically con-

stricted to 3.5 μm (average difference of 3.2 μmbetween

maximum width and width of the tips); subterminal cells

and the cells below usually shorter and gradually wider,

subterminal cells occasionally branched. Hyphal termina-

tions near the pileus centre slightly slender and apically

less attenuated; terminal cells slightly shorter (40–)51.8–

72.9–94.0(−124) × (4–)4.5–6.0–7.5(−10) μm, subterminal

cells never branched. Pileocystidia near the pileus margin

widely dispersed, 1–3 celled, long, terminal cells

(61–)73.0–97.5–122.0(−160) × (5–)5.5–7.0–8.5(−10) μm,

cylindrical to subulate, flexuose, sometimes with small

lateral projection, apically obtuse or with 1–2 eccen-

tric appendages, sometimes bifurcating, with oily gut-

tulate content, without reaction in sulfovanillin; near

the pileus centre widely dispersed, 1–2 celled, gener-

ally shorter, terminal cells (52–)58.0–75.2–92.4(−

115) × (4–)5.1–6.8–8.5(−11) μm, similar in shape and

content, mostly apically with 1–2 eccentric append-

ages, not bifurcating. Pileocystidia not near surface

but only deeper in the pileipellis. Oleiferous hyphae

containing brown pigments and cystidioid hyphae

present in the trama.

Ecology: Growing with Fagus sylvatica,Abies alba,

Picea abies, and Carpinus betulus.

Distribution: Known from Austria, the Czech Republic,

Estonia, Germany, Norway, and Slovakia.

Fig. 6 Russula albonigra (SAV F-20177), pileocystidia. aNear the

pileus margin. bNear the pileus centre. Bar = 10 μm

De Lange et al. IMA Fungus (2021) 12:20 Page 12 of 31

Additional material studied:Slovakia:Stužica Natural

reserve, under Kýčera hill, with Abies and Fagus, 22 Sept.

2017, S. Adamčík, (SAV F-20177); Stužica National Nature

Reserve, central part, with Fagus sylvatica, 5 Oct. 2003, S.

Adamčík, (SAV F-755); Badínsky prales Nature reserve,

with Abies and Fagus, 22 Sept. 2017, S. Adamčík,(SAVF-

2559); Badínsky prales Nature reserve, with Abies and

Fagus, 29 Sept. 2011, S. Adamčík, (SAV F-3465); Revúca,

road to Sirk, with Fagus sylvatica, 14 Oct. 2015, S. Adam-

čík, (SAV F-4776). –Germany: Bavaria, Oberallgäu, Ober-

staufen, Hündle, alt. 975 m, N47°32′45.9″E10°04′15.1″,

with Abies alba,Fagus sylvatica and Picea abies, 6 Sept.

2013, J. Kleine, JK RUS 13090603 (hb. Jesko Kleine). –

Czech Republic: Central Bohemia, Chrudim District,

Bojanov (Horní Bezděkov), under Fagus, 12 Aug. 2012, J.

Borovička, (PRM 934322); Central Bohemia, Kladno

District, Běleč(Jenčov), under Fagus and Carpinus,3Sept.

2013, J. Borovička, (PRM 924409).

Notes:Russula albonigra was first described by

Krombholz (1845)asAgaricus alboniger with only a

brief description and no holotype designated. Later, Fries

classified it in the genus Russula under its current name

(Fries 1874) that became well-known and widely used in

Europe. The illustration (Krombholz 1845: pl. 70, Figs. 16

and 17; Biodiversity Heritage Library), reproduced in

Fig. 7and made by Krombholz was suggested to serve as

lectotype by Sarnari, although it was never formally des-

ignated (Sarnari 1998). The illustration is the only avail-

able original material and is hereby formally designated

as the lectotype. The brief description and the plate itself

are not sufficient to determine which species of the

R. albonigra complex corresponds to R.albonigra.

This makes the collecting area of Krombholz the

most relevant criterion and in Krombholz (1845)itis

mentioned that R.albonigra is found in Prague.

Within the dataset, collection PRM 924409 was found

Fig. 7 Krombholz (1845). Image from the BHL (Biodiversity Heritage Library). Contributed by Missouri Botanical Garden, Peter H. Raven Library

De Lange et al. IMA Fungus (2021) 12:20 Page 13 of 31

only 31 km from the Prague city centre. Therefore,

the clade in which this collection is placed, is chosen

to represent R.albonigra. Specimen SAV F-20197 is

here designated as epitype.

Russula ambusta De Lange, Adamčík & F. Hampe,

sp. nov. (Figs. 3g, 8,9and 10).

MycoBank: MB839080.

Etymology: Refers to the appearance of the basidio-

mata, which look like they were burnt.

Diagnosis: Differs from the other species of the

Russula albonigra complex by the intermediate

reticulation and density of spore ornamentation and

the presence of appendages, but lack of bifurcations

on the pileocystidia.

Type:Slovakia: Vývrať,Bučková, W slopes of the hill,

with Quercus, 6 July 2011, V. Kučera (SAV F-3558 –

holotype).

Description: Pileus large, 45–100 mm diam, planocon-

vex to applanate, centrally depressed to umbilicate,

becoming more infundibuliform when older; margin

slightly inflexed when young, straight when mature,

smooth; pileus surface smooth, dry, dull to somewhat

viscid when wet; greyish orange, light brown (5B5, 5D5)

Fig. 8 Russula ambusta (SAV F-3358, FH 2008 ST01), hymenium. aBasidia. bMarginal cells. cBasidiospores. dCystidia near lamellae edges.

eCystidia on lamellae sides. Bar = 10 μm, except for c 5 μm

De Lange et al. IMA Fungus (2021) 12:20 Page 14 of 31

to umber, greyish brown (5F8, 6F3) with paler white to

sand coloured patches. Lamellae segmentiform to sub-

ventricose, to 6 mm deep, adnate to subdecurrent; snow

white, later yellowish white (4A2), blackening with age

or when bruised; with numerous lamellulae of different

lengths; dense (7–10 L + 5–8 l/cm at mid-radius); edges

even, concolorous, blackening with age. Stipe 30–40 ×

15–25 mm, cylindrical, firm and fleshy, smooth; white

but rapidly almost completely orange brown; solid in-

side. Context ca. 3–5 mm thick at mid-radius, firm,

white, greying before blackening, no reddening observed;

turning immediately dark blue with guaiac (strong reac-

tion, +++); taste first mild, then quickly somewhat cool-

ing with menthol component, never spicy or acrid;

odour indistinct. Spore print white (Ia).

Fig. 9 Russula ambusta (SAV F-3358, FH 2008 ST01), hyphal

terminations of the pileipellis. aNear the pileus margin. bNear the

pileus centre. Bar = 10 μm

Fig. 10 Russula ambusta (SAV F-3358), pileocystidia. aNear the

pileus margin. bNear the pileus centre. Bar = 10 μm

De Lange et al. IMA Fungus (2021) 12:20 Page 15 of 31

Basidiospores (6.7–)7.4–8.2–9.0(−9.6) × (5.4–)5.7–6.0–

6.3(−6.8) μm, broadly ellipsoid to narrowly ellipsoid, Q =

(1.18–)1.28–1.38–1.48(−1.54); ornamentation of very low,

dense to very dense [(6–)7–13(−16) in a 3 μmdiamcircle]

amyloid warts, up to 0.2 μm high, subreticulate to reticulate,

abundantly fused in chains [(2–)3–8(−10) fusions in a 3 μm

diam circle], and also connected by short, fine line connec-

tions [(0–)1–9(−17) in a 3 μm diam circle]; suprahilar spot

medium-sized, not amyloid. Basidia (54–)57.5–62.6–67.7(−

76) × (8–)8.9–9.5–10.1(−11) μm, narrowly clavate, 4-spored.

Hymenial cystidia (66–)67.1–91.9–107.7(−125) × (7–)8.5–

9.8–11.1(−13) μm, variable: (1) narrowly fusiform to

narrowly clavate, flexuose to even slightly moniliform,

apically obtuse or with a constriction or with small

appendage to even slightly mucronate, thin-walled;

with heteromorphous, oily content, mostly fragmented

in multiple crystalline-like masses or slightly granu-

lose, without reaction in sulfovanillin; (2) narrowly

fusiform to lanceolate, flexuose, tapering towards the

top; with less content, heteromorphous, oily, mostly

fragmented in multiple crystalline-like masses or

slightly granulose, without reaction in sulfovanillin; near

the lamellae edges, (29–)37.0–63.7–90.4(−144) × (5–)7.2–

8.5–9.8(−10) μm, (1) narrowly fusiform to narrowly cla-

vate, flexuose, apically obtuse or tapering towards the top

in a moniliform way with a small appendage, thin-walled;

content as on lamellae sides; (2) as on lamellae sides.

Lamellae edges sterile, when older elements can con-

tain brown pigments; marginal cells (15–)19.1–26.1–

33.1(−36) × (4–)4.7–5.6–6.5(−7) μm, undifferentiated,

cylindrical to narrowly clavate, thin-walled. Pileipellis

orthochromatic in Cresyl Blue, 175–400 μm deep, not

sharply delimited from trama, gradually passing; sub-

pellis not delimited from suprapellis; hyphae 3–6μm

wide near trama, dense near surface and near trama,

irregularly oriented, more parallel and horizontal near

trama and surface, intricate everywhere, pigmented

near surface only, with no distinct gelatinous coating.

Acid-resistant incrustations absent. Hyphal termina-

tions near the pileus margin long, with multiple septa,

flexuous, thin-walled, filled with irregular refractive

bodies containing brown pigments; terminal cells

(41–)51.4–70.2–89.0(−125) × (3–)4.3–5.4–6.5(−8) μm,

narrowly cylindrical, on average apically constricted to

3.5 μm; subterminal cells and the cells below similar

in length or slightly shorter, similar in width or grad-

ually slightly wider, subterminal cells and cells below

sometimes branched. Hyphal terminations near the

pileus centre similar, terminal cells usually shorter

(28–)40.8–59.8–78.8(−106) × (3–)4.0–5.0–6.0(−7) μm,

subterminal cells and cells below not branching.

Pileocystidia hard to find; near the pileus margin

widely dispersed to rare, mostly 1-celled, but up to 3-

celled, long, terminal cells (73–)79.1–110.6–142.1(−

145) × 4.9–7.2–9.5(−11) μm, cylindrical, flexuose, apically

mostly with 2–3 eccentric appendages or obtuse, content as

in hymenial cystidia or more granulose, without reaction in

sulfovanillin; near the pileus centre widely dispersed, 1-

celled, (63–)79.4–106.7–134.0(−151) × (4–)5.0–6.4–7.8(−

8) μm, cylindrical to slightly subulate, apically with double

constriction or 1–2 eccentrical appendages, content as near

pileus margin. Oleiferous hyphae containing brown pig-

ments and cystidioid hyphae present in the trama.

Ecology: Growing with Quercus robur, Pinus sylvestris,

and Betula pendula.

Distribution: Known from Estonia, Germany, Slovakia,

Spain, and Sweden.

Additional material studied:Germany:Brandenburg,

Landkreis Oder-Spree, near Helenesee Frankfurt-Oder,

Markendorfer Forst, MTB 3752/2 (Müllrose), N 52.256386

E 14.46049, lichen-pine (Pinus sylvestris) forest with inter-

spersed birch trees (Betula pendula)onanunpaved

forest path, on sandy soil, 19 Oct. 2008, F. Hampe &

I. Kindermann, FH 2008 ST01 (hb. Felix Hampe).

Notes: The range of the spore size is large within this

species. This results from the sporesizedifferencebetween

the collections. The holotype (SAV F-3558) has smaller

spores and lower Q-value than collection FH 2008 ST01.

The variability in the shape of the hymenial cystidia is also

due to the difference between the collections with the holo-

type having cystidia of type 1 and collection FH 2008 ST01

having cystidia of type 2 (types referring to (1) and (2) in

the description). Therefore, based on morphology, we

could hypothesize that these collections represent different

species. Nevertheless, we treat them here as the same

species because phylogenetically there is no support for the

hypothesis of two different species. All markers used in this

study place these two collections together as the same

species. Measurements are given for each collection

separately in Supplementary Material 1. Of course, more

collections and more micromorphological study are

needed in order to understand this intraspecific variation.

The UNITE species hypothesis at 0.5% corresponding to

our concept of R. ambusta is based on a locked sequence

and after this publication we will propose to change the

reference sequence to the holotype of the species.

Russula nigrifacta De Lange & Adamčík, sp. nov.

(Figs. 3a-b, 11,12 and 13).

MycoBank: MB839081.

Etymology: Named after the strong blackening of the

basidiomata.

Diagnosis: Differs from the other species of the

Russula albonigra complex by the lack of both appendages

and bifurcations on the pileocystidia.

Type:Italy: Tuscany, Province of Livorno, Piombino,

with Quercus ilex and Quercus suber, 9 Nov. 2016, R. De

Lange, RDL 16–044 (GENT –holotype).

De Lange et al. IMA Fungus (2021) 12:20 Page 16 of 31

Description:Pileus large, 75–105 mm diam, planocon-

vex to applanate, centrally depressed to umbilicate, be-

coming more infundibuliform when older; margin slightly

inflexed when young, straight when mature, smooth; pi-

leus surface smooth, sometimes slightly cracked at the

margin, dry, dull to somewhat viscid when wet; ivory to

cream, sand coloured, yellowish white (4A2, 5A3, 5B3)

with darker spots of pale brownish/greyish orange, yellow-

ish brown to light brown, dark brown (5B6, 5E4, 5E8,

5F4). Lamellae narrow, segmentiform to subventri-

cose, 2–4 mm deep, adnate to subdecurrent; white to

yellowish white, rarely with faintly blueish shine,

blackening with age; with numerous lamellulae of different

lengths without clear regular pattern, rarely locally anasto-

mosing; dense (6–9L+8–9l/cm at mid-radius); edges

even, concolorous, blackening with age. Stipe 30–60 × 19–

30 mm, cylindrical, firm and fleshy, smooth; white, becom-

ing more orange brown with age; solid inside. Context ca.

6–7 mm thick at mid-radius, firm, white, blackening with-

out reddening, surface of pileus and stipe also slightly red-

dening before blackening; turning greenish with FeSO

yellowish with KOH, immediately dark blue with guaiac

(strong reaction, +++); taste mild to slightly refreshing;

odour fruity, sweet. Spore print white (Ia).

Fig. 11 Russula nigrifacta (RDL 16–028, RDL 16–044/2, RDL 16–063, SAV F-2418), hymenium. aBasidia. bMarginal cells. cBasidiospores. dCystidia

near lamellae edges. eCystidia on lamellae sides. Bar = 10 μm, except for c 5 μm

De Lange et al. IMA Fungus (2021) 12:20 Page 17 of 31

Basidiospores (6.7–)7.5–8.0–8.5(−9.5) × (5.0–)5.7–6.0–

6.3(−7.0) μm, broadly ellipsoid to ellipsoid, Q =

(1.15–)1.25–1.34–1.43(−1.56); ornamentation of low, dense

[(5–)7–10(−11) in a 3 μm diam circle] amyloid warts, 0.2–

0.4 μm high, subreticulate, abundantly fused into chains

[(0–)3–6(−8) fusions in a 3 μm diam circle] and also con-

nected by short, fine line connections [0–3(−4) in a

3μm diam circle]; suprahilar spot small, not amyloi d.

Basidia (50–)57.0–62.8–68.5(−79) × (8–)8.7–9.6–

10.5(−11) μm, narrowly clavate, 4-spored. Hymeni al cystidia

(57–)62.6–82.6–102.6(−128) × (7–)7.7–8.9–10.1(−15) μm,

cylindrical to narrowly fusiform to narrowly clavate, some-

times slightly flexuose, apically obtuse or with small ap-

pendage, thin-walled; with heteromorphous, oily content,

fragmented in multiple crystalline-like masses, without

clear reaction in sulfovanillin; near the lamellae edges,

(37–)53.0–69.4–85.8(−119) × (7–)8.0–9.1–10.2(−11) μm,

cylindrical to narrowly fusiform, sometimes narrowly

clavate, often slightly flexuose, apically obtuse with small

appendage or mucronate to tapering towards the top,

thin-walled; content as on lamellae sides. Lamellae edges

sterile, when older elements can contain brown pigments;

Fig. 12 Russula nigrifacta (RDL 16–028, RDL 16–044/2, RDL 16–063, SAV F-2418, SAV F-2419), hyphal terminations of the pileipellis. aNear the

pileus margin. bNear the pileus centre. Bar = 10 μm

De Lange et al. IMA Fungus (2021) 12:20 Page 18 of 31

marginal cells (15–)17.7–22.2–26.7(−30) × (4–)4.6–5.9–

7.2(−10) μm, poorly differentiated, cylindrical to narrowly

clavate, flexuose, thin-walled. Pileipellis orthochromatic in

Cresyl Blue, 90–136 μm deep, not sharply delimited from

trama and not gradually passing, intermediate; subpellis

not well delimited from suprapellis; hyphae 2.5–7μmwide

near trama, dense near surface and near trama, irregularly

oriented, more parallel and horizontal near trama, pigmen-

ted throughout the pileipellis, with some gelatinous coating.

Acid-resistant incrustations absent. Hyphal terminations

near the pileus margin long, slender, with multiple septa,

flexuous, thin-walled, filled with irregular refractive bodies

containing brown pigments; terminal cells (50–)63.5–83.5–

103.5(−133) × (3–)4.0–4.9–5.8(−7) μm, narrowly cylin-

drical; subterminal cells and the cells below similar in

length and width, subterminal cells never branched.

Hyphal terminations near the pileus centre similar;

terminal cells (42–)53.1–79.6–106.1(−135) × (3–)3.4–

4.6–5.8(−8) μm, sometimes more subulate, subter-

minal cells and cells below shorter, subterminal cells

rarely branched. Pileocystidia near the pileus margin

dispersed, 1–4 celled, very long, terminal cells

Fig. 13 Russula nigrifacta (RDL 16–028, RDL 16–044/2, RDL 16–063, SAV F-2418, SAV- F-2419), pileocystidia. aNear the pileus margin. bNear the

pileus centre. Bar = 10 μm

De Lange et al. IMA Fungus (2021) 12:20 Page 19 of 31

(41–)83.4–119.5–155.6 (−235) × (5–)5.8–6.7–7.6(−9) μm, cy-

lindrical, flexuose, apically obtuse or with slight constriction,

with oily granulose content, without clear reaction in sulfova-

nillin; near the pileus centre dispersed, 1–2 celled, similar in

shape and content or with less and more oily guttulate con-

tent, terminal cells (64–)80.9–123.0–165.1(−220) × (4–)5.2–

6.0–6.8(−7) μm. Oleiferous hyphae containing brown pig-

ments and cystidioid hyphae present in the trama.

Ecology: Growing with Mediterranean oaks (Quercus

ilex and Quercus suber) in Italy, in Slovakia with Quercus

robur, and Carpinus betulus.

Distribution: Known from Estonia, Italy, and Slovakia.

Additional material studied:Italy: Tuscany, Province of

Livorno, Piombino, with Quercus ilex and Quercus suber,7

Nov. 2016, R. De Lange,RDL16–028 (GENT); Tuscany, Prov-

ince of Livorno, Piombino, with Quercus ilex and Quercus

suber, 11 Nov. 2016, R. De Lange,RDL16–063 (GENT). –

Slovakia:Obyce,forestNEofthevillage,withQuercus and

Carpinus, 24 July 2008, S. Adamčík, (SAV F-2418); Prenčov,

Horné Majere, with Quercus and Carpinus, 22 July 2008,

S. Adamčík, (SAV F-2419); T e pličky, with Quercus

and Carpinus, 4 July 2009, S. Adamčík, (SAV F-3006);

Bohunický Roháč, forest close to the nature reserve, with

Quercus,8Sept.2006,S. Adamčík, (SAV F-1501).

Fig. 14 Russula ustulata (AV 16–019), hymenium. aBasidia. bMarginal cells. cBasidiospores. dCystidia near lamellae edges. eCystidia on lamellae

sides. Bar = 10 μm, except for c 5 μm

De Lange et al. IMA Fungus (2021) 12:20 Page 20 of 31

Notes: Besides the diagnostic features mentioned in the diag-

nosis, R. nigrifacta is characterised by its higher spore orna-

mentation (incomplete reticulum) and the thin pileipellis.

These features are shared with R.albonigra,whichis

easily differentiated from the other species in the

complex by the oily guttulate content of the cystidia.

Furthermore, our data suggests that R.nigrifacta is associ-

ated with Quercus spp. (and possibly also with Carpinus

betulus) in thermophilous habitats (thermophilous oak

forests and Mediterranean oak forests).

Russula ustulata De Lange & Verbeken, sp. nov. (Figs.

3h, 14 and 15).

MycoBank: MB839082.

Etymology: Refers to the appearance of the basidio-

mata, which look like they were burnt.

Diagnosis: Differs from the other species of the Russula albo-

nigra complex by the absence (or rareness) of pileocystidia.

Type:Norway: NT Steinkjer, Kvamsfjellet, North of Lyst-

jörna, Austerolsenget, alt. 137.5 m, N64°12′47″E11°49′09″,

20 Aug. 2016, A. Verbeken,AV16–019 (GENT –holotype).

Description:Pileus large, 101–112 mm diam, planoconvex

to applanate, centrally depressed to umbilicate to infundibu-

liform, widely V-shaped; margin straight, smooth; pileus sur-

face smooth, glabrous, dry, shiny; very dark blackish brown

to dark grey-black (7F2–4) without lighter brown tints, uni-

form. Lamellae narrow, segmentiform to subventricose, 4–5

mm deep, adnate to subdecurrent; completely white to

Fig. 15 Russula ustulata (AV 16–019), pileipellis. aHyphal terminations near the pileus margin. bHyphal terminations near the pileus centre.

cPileocystidia near the pileus margin. Bar = 10 μm

De Lange et al. IMA Fungus (2021) 12:20 Page 21 of 31

yellowish white, quickly blackening; with numerous

lamellulae of different lengths, locally anastomosing; mod-

erately distant (6–9L+3–4 l/cm at mid-radius) to distant

(sometimes almost as in R. nigricans); edges even, papery

thin, concolorous, quickly blackening with age. Stipe 45–

65 × 25–30 mm, cylindrical, firm and fleshy, smooth to

irregular surface, dry; white but rapidly turning dark grey,

black (stays whitish only at the top); solid inside. Context

ca. 5–9 mm thick at mid-radius, firm, white, staining grey,

then brownish black, mostly blackening without redden-

ing but a slight pink tinge can be present; turning greenish

with FeSO

; taste mild but not agreeable, musty, but

slightly menthol-like in the gills. Spore print white (Ia).

Basidiospores (7.4–)8.1–8.5–8.9(−9.3) × (5.7–)6.0–6.3–

6.6(−6.9) μm, broadly ellipsoid to ellipsoid, Q =

(1.12–)1.26–1.36–1.46(−1.48); ornamentation of very

low, very dense [(9–)10–15(−17) in a 3 μm diam circle]

amyloid warts, to 0.2 μm high, reticulate, abundantly

fused into chains [3–7(−10) fusions in a 3 μm diam

circle], abundantly connected by short, fine line connec-

tions [(6–)7–11(−13) in a 3 μm diam circle]; suprahilar

spot large, not amyloid. Basidia (53–)58.9–67.3–75.7(−

83) × (8–)8.4–9.2–10.0(−11) μm, narrowly clavate to cy-

lindrical, 4-spored. Hymenial cystidia (78–)88.7–106.6–

124.5(−153) × (7–)8.0–9.1–10.2(−12) μm, cylindrical to

narrowly fusiform, apically obtuse with small appendage

or slightly tapering towards the top, thin-walled; with

heteromorphous, oily content, fragmented in multiple

masses to needle-like crystalline, without reaction in sulfo-

vanillin; near the lamellae edges, (60–)70.0–82.9–95.8(−

111) × (7–)8.1–9.5–10.9(−14) μm, narrowly fusiform to

narrowly conical, slightly flexuose, apically obtuse, some-

times with small appendage or tapering towards the top,

thin-walled; content as on lamellae sides, often containing

brown pigments. Lamellae edges sterile, when older ele-

ments can contain brown pigments; marginal cells

(14–)19.8–26.5–33.2(−39) × 4.9–6.3–7.7(−10) μm, poorly

differentiated, cylindrical to narrowly clavate to fusiform,

flexuose, thin-walled. Pileipellis orthochromatic in Cresyl

Blue, 250–300 μm deep, not sharply delimited, gradually

passing; subpellis not delimited from suprapellis; hyphae

3–7μm wide near trama, dense near surface and near

trama, loose in intermediate zone, irregularly oriented,

pigmented throughout the pileipellis, some gelatinous

coating can be present deeper in the pileipellis. Acid-resist-

ant incrustations absent. Hyphal terminations near the

pileus margin long, with multiple septa, flexuous, thin-

walled, filled with irregular refractive bodies containing

brown pigments; terminal cells (29–)36.0–53.6–71.1(−

100) × (4–)4.9–6.0–7.1(−9) μm, narrowly cylindrical to

subulate, on average apically constricted to 4.5 μm; subter-

minal cells and the cells below shorter and gradually

wider, subterminal cells never branched, cells below occa-

sionally branched. Hyphal terminations near the pileus

centre slightly slender and apically less attenuated;

terminal cells slightly longer, (31–)43.3–59.0–74.7(−

89) × (3–)4.0–5.1–6.2(−8) μm, subterminal cells and cells

below more of similar size as terminal cells, subterminal

cells rarely branched. Pileocystidia near the pileus margin

extremely rare (only 5 observed), inconspicuous, hardly

distinguishable, 58.2–79.6–101.0(−115) × (6–)6.3–7.0–

7.7(−8) μm, cylindrical to narrowly clavate, apically ob-

tuse, content as hymenial cystidia but very little, near the

pileus centre absent. Oleiferous hyphae containing brown

pigments present in the trama, cystidioid hyphae absent.

Ecology: Growing with Picea abies, and Pinus sylvestris.

Distribution: Known from the Czech Republic, Estonia,

Finland, Italy, Norway, and the Russian Federation.

Additional material studied:Italy: Langhestel, with Picea and

Pinus sylvestris, 25 Sept. 1997, S. Adamčík, (SAV F-2610). –

Czech Republic: South Bohemia, Český Krumlov district,

Malonty, with Picea and Pinus sylvestris,togetherwithTricholoma

matsutake, 2 Sept. 2014, J. Borovička, (PRM 924452).

Notes: Our data suggests that Russula ustulata has a

specific ecology, different from the other species in the

complex. It is associated with coniferous trees in boreal

forests or mountain habitats.

Russula sp. 1 (Figs. 16,17 and 18).

Description:Pileus large, planoconvex to applanate,

centrally depressed to umbilicate to infundibuliform;

margin straight, smooth; pileus surface smooth, dry, dull;

light brown to greyish brown, dark brown, with paler

white to sand coloured patches. Lamellae narrow, seg-

mentiform to subventricose, adnate to subdecurrent;

completely white to yellowish white, quickly blackening;

with numerous lamellulae of different lengths; moder-

ately distant; edges even, concolorous, quickly blacken-

ing with age. Stipe cylindrical, firm and fleshy, smooth to

irregular surface, dry; white but turning orange brown to

dark grey, black; solid inside. Context firm, white, blacken-

ing without reddening; taste mild. Spore print white (Ia).

Basidiospores (7.4–)7.6–7.9–8.2(−8.4) × (6.0–)6.1–6.3–

6.5(−6.6) μm, broadly ellipsoid, Q = (1.18–)1.22–1.26–

1.30(−1.33); ornamentation of very low, very dense

[(8–)9–15(−16) in a 3 μm diam circle] amyloid warts, to

0.3 μm high, reticulate, abundantly fused into chains

[(1–)2–7(−11) fusions in a 3 μm diam circle],

abundantly connected by short, fine line connections

[(5–)7–13(−15) in a 3 μm diam circle]; suprahilar spot

medium-sized, not amyloid. Basidia (54–)56.7–60.9–65.1(−

69) × (9–)9.4–9.9–10.4(−11) μm, narrowly clavate, 4-spored.

Hymenial cystidia,64.0–83.6–103.2(−130) × (7–)7.9–8.7–

9.5(−10) μm, cylindrical to narrowly fusiform, often slightly

moniliform and flexuose, apically obtuse or with double con-

striction or small appendage, thin-walled; with heteromorph-

ous, oily content, fragmented in multiple crystalline-like

masses, without reaction in sulfovanillin; near the lamellae

De Lange et al. IMA Fungus (2021) 12:20 Page 22 of 31

edges, (53–)56.2–62.3–68.4(−76) × (6–)6.9–7.8–8.7(−9) μm,

cylindrical to narrowly fusiform, often slightly moniliform

and flexuose, apically obtuse or with small appendage, thin-

walled; content as on lamellae sides. Lamellae edges sterile,

elements containing brown pigment when older; marginal

cells (15–)16.3–21.5–26.7(−30) × (5–)5.3–6.6–7.9(−9) μm,

undifferentiated, cylindrical to narrowly clavate, thin-walled.

Pileipellis orthochromatic in Cresyl Blue, 200–275 μm deep,

not sharply delimited from trama and not gradually passing,

intermediate; subpellis not delimited from suprapellis; hy-

phae 3–5μmwideneartrama,moredensenearsurfaceand

near trama, irregularly oriented, more parallel and horizontal

near trama, pigmented only in the upper part of the pileipel-

lis, with some gelatinous coating. Acid-resistant incrustations

absent. Hyphal terminations near the pileus margin long,

with multiple septa, scarcely branched at the bases, flexuous,

thin-walled, filled with irregular refractive bodies containing

brown pigments; terminal cells (39–)47.6–61.4–75.2(−86) ×

5.0–6.0–7.0(−8) μm, narrowly cylindrical to slightly subulate

or narrowly fusiform, on average apically constricted to

4.5 μm; subterminal cells and the cells below similar in length

and width or slightly wider, subterminal cells never branched.

Hyphal terminations near the pileus centre slightly wider and

apically more attenuated, containing inflated cells, more

Fig. 16 Russula sp. 1 (RW 1975), hymenium. aBasidia. bMarginal cells. cBasidiospores. dCystidia near lamellae edges. eCystidia on lamellae

sides. Bar = 10 μm, except for c 5 μm

De Lange et al. IMA Fungus (2021) 12:20 Page 23 of 31

Fig. 18 Russula sp. 1 (RW 1975), pileocystidia. aNear the pileus

margin. bNear the pileus centre. Bar = 10 μm

Fig. 17 Russula sp. 1 (RW 1975), hyphal terminations of the pileipellis.

aNear the pileus margin. bNear the pileus centre. Bar = 10 μm

De Lange et al. IMA Fungus (2021) 12:20 Page 24 of 31

often branched at the bases; terminal cells (28–)46.3–70.5–

94.7(−130) × (3–)3.9–5.2–6.5(−8) μm, subterminal cells

rarely branched. Pileocystidia near the pileus margin numer-

ous, 1 celled, rarely 2 celled, extremely long, terminal cells

98–more than 320 × (8–)8.2–9.1–10.0 μm, cylindrical, flex-

uose, apically obtuse or with slight constriction, sometimes

bifurcating, content as in hymenial cystidia or more granu-

lose, without reaction in sulfovanillin; near the pileus centre

numerous, 1–2 celled, generally shorter but still very long,

terminal cells 56.6–117.4–178.2(−298) × (5–)6.1–8.4–10.7(−

14) μm, similar in shape and content, rarely with up to 3

eccentric appendages. Oleiferous hyphae containing brown

pigments and cystidioid hyphae present in the trama.

Ecology: Growing with Mediterranean cork oak (Quercus

suber).

Distribution: Known from Italy (Sardinia).

Specimen examined:Italy: Sardinia, Tempio Pausania,

road SS133, between 3 and 4 km from centre, with Quercus

suber, 1 Nov. 2000, R. Walleyn,RW1975(GENT).

Notes: Although there is molecular and morphological

support that this collection represents a species different

from the other species in the Russula albonigra complex,

the authors chose to not formally describe this

Key to the European species of Russula subgen. Compactae

1 Height of spore ornamentation not exceeding 0.5 μm .......................................................... 2

Height of spore ornamentation exceeding 0.5 μm ....................................... roseonigra

2 (1) Content of hymenial cystidia not reacting in sulfovanillin ..................................................... 3

Content of hymenial cystidia at least greying in sulfovanillin ...................................... 8

3 (2) Basidiomata sturdy and fleshy; lamellae thick and very distant; context quickly and strongly reddening

before Lamellae thin and moderately distant; context not reddening before blackening or only slightly at the

surface; pileocystidia often exceeding 90 μm ................................ 4

4 (3) Content of all cystidia oily guttulate ........................................................................ albonigra

Content of cystidia not oily guttulate .......................................................................... 5

5 (4) Pileocystidia absent or extremely rare and only near the pileus margin ..................... ustulata

Pileocystidia at least widely dispersed and found throughout the whole surface of the pileus

......................................................................................................................... 6

6 (5) Pileocystidia never with appendages or bifurcations ............................................... nigrifacta

Pileocystidia (at least some) containing appendages or bifurcations ........................... 7

7 (6) Pileocystidia numerous, extremely long (some exceeding 200 μm), with both appendages and bifurcations

present; hyphal terminations of the pileipellis with inflated cells ................ sp. 1

Pileocystidia widely dispersed, never exceeding 160 μm, lacking bifurcations; hyphal terminations of the

pileipellis without inflated cells ....................................... ambusta

8 (2) Taste mild ............................................................................................................................ 9

Taste, at least in the lamellae, noticeably acrid......................................................... 10

9 (8) Context not clearly reddening but quickly and strongly blackening, sometimes with a slightmenthol-like

taste Context turning slowly pale pink before greying (but almost simultaneously, and without strong

blackening); smell of old wine barrels; pileocystidia present; spore ornamentation not exceeding 0.2

μm................................................................ adusta

10 (8) Hyphal terminations of the pileipellis consisting of shorter, broad, swollen cells ................ 11

Hyphal terminations of the pileipellis consisting of longer, slender cells ................... 12

11 (10) Context clearly reddening before turning grey-black .............................................. densifolia

Context not clearly reddening, first greying then blackening....................... densissima

12 (10) Context quickly and strongly blackening; lamellae with a distinct pinkish shine; pileocystidiaabsent;

hyphal terminations Context not so strongly and quickly blackening, more greying or slowly blackening;

pileocystidia present ................................................................................................ 13

13 (12) Context strongly reddening, then greying; taste burning acrid, in the flesh as well as in the lamellae

acrifolia Context not clearly reddening, slowly blackening; taste acrid, but not burning and more prominent

in the lamellae than in the context..................................... fuliginosa

De Lange et al. IMA Fungus (2021) 12:20 Page 25 of 31

species here as the description is based on a single

collection only.

DISCUSSION

Our study recognises five species within the traditional

concept of Russula albonigra and this name was the only

available at the species rank. Before new names could be

assigned to undescribed species, identification of the old

name proofed to be challenging, especially because it

was already used in the early Friesian period and its ori-

ginal description (Krombholz 1845) does not meet

current morphological standards and does not provide

sufficient detail. The species clade identified in this study

as R. albonigra has the best match with the probable

geographic and ecological origin of the species described

by Krombholz.

The traditional morphological concept of R. albonigra

(Romagnesi 1967; Sarnari 1998; Kibby 2001) more or

less agrees with our observations of the species complex.

We think that in the field, for the preliminary identifica-

tion of species to the R. albonigra complex, the following

characters are most helpful: a strong and fast blackening

of the surface resulting in a distinct contrast of wounded

compared to untouched areas, the relatively sturdy and

thick-fleshed basidiomata, a mild or refreshing taste of

the context, moderately distant and strongly blackening

lamellae and a dry, usually dull and not viscid pileus cu-

ticle. However, macroscopically the species within the R.

albonigra species complex are very alike and cannot be

distinguished unambiguously. Nonetheless, we observed

that the reaction to FeSO

could be interesting to differ-

entiate some of the species. While R.albonigra has an

orange reaction to FeSO

, both R.nigrifacta and R.ustu-

lata have a greenish reaction. As this data is missing for

R.ambusta and R. sp. 1, it is important to pay attention

to this character in future collections. There are some

interesting observations questioning the traditional char-

acteristics used to define the R.albonigra complex. First

of all, it seems that the absolute lack of any reddening is

not a reliable feature, because it can be weak and easily

overseen or vanishing quickly due to the strong and

quick blackening. At least three of the species within the

R.albonigra complex (R.albonigra, R.nigrifacta, and R.

ustulata) comprise a collection where some weak red-

dening is observed at the surface or even of the context.

Some variability about the reddening reaction was also

noted by Romagnesi, who recognised Russula albonigra

f. pseudonigricans with an intense reddening context

(Romagnesi 1962; Romagnesi 1967). Attempts to get a

sequence from the type material failed. The holotype of

this form is in a bad condition which does not allow

good microscopic observations. Furthermore, a micro-

scopic study of a paratype suggested that holotype and

paratype did not represent the same species. We suggest

for now, until molecular data becomes available, not to

draw any conclusions about the identity of R.albonigra

f. pseudonigricans or its classification within the R.albo-

nigra complex. Moreover, names described in our study

have priority at species rank over any future combin-

ation of the f. pseudonigricans epithet (Art. 11.2 of the

ICNafp). Although we believe that the reddening reac-

tion of the context can be used as a diagnostic between

species on opposite sides of the spectrum (i.e. species

with a strong reddening reaction versus species without

a clear reaction) some caution is needed.

Another traditional morphological character to define

R. albonigra was the characteristic menthol taste in the

lamellae. This does not seem to be a stable character be-

cause it was not observed in all collections and it can

also depend on the subjective opinion of an individual

person. Furthermore, this menthol-refreshing taste is

also noted to possibly be present in R.atramentosa by

Sarnari (1998).

We found the lack of a reaction of the cystidial con-

tent to sulfovanillin to be a good synapomorphic charac-

ter to define the R. albonigra complex. Russula nigricans

is the only species outside this complex also showing no

clear reaction of the cystidial content to sulfovanillin.

But the latter species can easily be distinguished from

the R.albonigra complex by its thick and very distant la-

mellae, the strong reddening of the context and its pileo-

cystidia that are much shorter (never exceeding 90 μm).

Our conclusions about the delimitation of the species

complex are only based on observations of European

taxa of R. subgen. Compactae.

The second challenge of this study was to define mor-

phological differences among the species of the R. albo-

nigra complex defined by phylogenetic analyses. Due to

the low morphological variability we could consider the

species within the R.albonigra complex pseudocryptic

species (i.e. species with a morphological resemblance

that seems indistinguishable at first, but can be distin-

guished when using the appropriate characters; Delgat

et al. 2019). This is a phenomenon that is widely distrib-

uted within the Russulaceae, especially in the genus Lac-

tifluus (Stubbe et al. 2010, Van de Putte et al. 2010, Van

de Putte 2012, De Crop et al. 2014, Van de Putte et al.

2016, Delgat et al. 2017, De Lange et al. 2018, Delgat

et al. 2019), but also within the genus Russula (Adamčík

et al. 2016a; Adamčík et al. 2016b; Caboňet al. 2019).

The most striking microscopical differences between the

species in the R. albonigra complex are the higher spore

ornamentation of R.nigrifacta and R.albonigra com-

pared to the other species within the complex. Russula

ustulata and R. sp. 1 have an almost complete and

denser reticulum than the incomplete reticulum of R.

nigrifacta and R.albonigra. The ornamentation of the

spores in R.ambusta is intermediate in reticulation and

De Lange et al. IMA Fungus (2021) 12:20 Page 26 of 31

density. R.albonigra is distinguishable by the unique oily

guttulate content of all cystidia. R.nigrifacta typically

lacks appendages and bifurcations on the pileocystidia

whereas these are present in R.albonigra and R. sp. 1.

Russula ambusta lacks bifurcations but appendages are

present. The most striking feature of R.ustulata is the

absence (or rareness) of pileocystidia, whereas R. sp. 1

has very long and numerous pileocystidia. The thickness

of the pileipellis is also an interesting character. R.albo-

nigra has the thinnest pileipellis followed by R.nigri-

facta, the other species in the complex have a much

thicker pileipellis. The presence of inflated subterminal

cells in the hyphal terminations of the pileipellis centre

is typical for R. sp. 1 and not observed in the other spe-

cies of the complex.

Collections used in this study often do not have pre-

cise ecological details to define ecological niches and

host tree preferences. However, habitat type and geo-

graphical data suggest biological relevance to recognise

closely related species (Ryberg 2015). Russula ambusta,

R. nigrifacta, and R. ustulata are closely related but seem

to inhabit ecologically different niches. Russula nigri-

facta occurs both with Mediterranean oaks (Quercus ilex

and Quercus suber) and Quercus robur and Carpinus

betulus in thermophilous oak forests. Possibly, Russula

sp. 1 has a similar ecology,it is only known from a single

collection associated with Mediterranean oak (Quercus

suber). Russula ustulata is up to now only known from

boreal or mountain habitats, associated with coniferous

trees (Picea sp., Pinus sp.). Russula ambusta and R.albo-

nigra seem to have a similar ecology and are associated

with a variety of trees in temperate to montane forest

types. Russula ambusta was collected with Quercus

robur,Pinus sylvestris, and Betula pendula;Russula

albonigra with Fagus sylvatica,Abies alba,Picea abies,

and Carpinus betulus.

Our study suggests that species within the Nigricanti-

nae clade have a limited area of distribution, unlike what

is often believed. All North American collections re-

trieved from GenBank and placed in the R. albonigra

complex are not clustered with the European ones and

probably represent different species (Fig. 2). The re-

trieved ITS data did not confirm that the distribution of

R. albonigra is not transcontinental (Singer 1958; Hesler

1961; Shaffer 1962; Kibby and Fatto 1990; Thiers 1994),

but rather it supports the hypothesis that none of the

European taxa within R. subgen. Compactae are present

in the United States (Adamčík and Buyck 2014). The

North American collections represent at least two differ-

ent species with a high macromorphological resemblance

to R.albonigra and they may represent R. sordida and R.

subsordida, both having a weak or negative reaction of the

pileocystidia to sulfovanillin (Adamčík and Buyck 2014).

This study shows that the R. albonigra complex is also

represented in Asia by a still undescribed Chinese species.

A multi-locus phylogeny resulting in a strong sup-

port of the European species within the R.albonigra

complex, stimulated the detailed search for morpho-

logical differences between the species. The species

described in this study are defined by integrated tax-

onomy combining multi-locus molecular data with

detailed morphological and ecological data (i.e. distri-

bution, climate, and host data).

Our study demonstrated that all species within the R.

albonigra complex, supported by the strict genealogic

concordance and coalescent-based species delimitation,

are strictly distinguished at the threshold of 99.5%, that

corresponds to a distance of 0.5% when performing a

UNITE search. Even a distance of 1% results in only two

UNITE species hypotheses both covering multiple phylo-

genetic species within the complex.This shows that there

is a low genetic diversity of the ITS region between the

species within this complex. A possible explanation for

this low genetic diversity is that the species within

the R.albonigra complex are only relatively recently di-

verged from each other, which could explain the relatively

short branch lengths (Fig. 2) and the low morphological

variability. The occupation of new habitats and the adap-

tation to new hosts could have caused the radiation seen.

This study shows that ITS sequence similarity thresh-

olds of 97% commonly used in metabarcoding studies

(Pauvert et al. 2019) are not sufficient to differentiate the

phylogenetically defined species of the R.albonigra com-

plex. This observation is also made for other species

complexes within the genus Russula (Adamčík et al.

2016b). The species thresholds retrieved in this study

agree with the conclusions of testing global fungal data-

bases as training datasets, that predicted the optimal

identity thresholds to discriminate filamentous fungal

species as 99.6% or 99.3% for ITS (Vu et al. 2019;Vu

et al. 2020). Badotti et al. (2017) rank Russula as the

genus with only 38% probability of correct identification,

but the study also ranks it into the group for which ITS

is a good marker while another Russulaceae genus, Lac-

tarius, is placed in a group for which ITS is a poor

marker. We do not promote the use of a universal

threshold value, but rather emphasise the importance of

searching for the best threshold value, according to the

fungal group of interest.

Our UNITE species hypothesis threshold testing meets

both major problems highlighted by Kõljalg et al. (2013):

(1) the lack of an inclusive, reliable public reference data

set; and (2) the lack of means to refer to fungal species,

for which no scientific name is available, in a standard-

ized stable way. First, our singleton collection of R. sp.1

is not represented in UNITE, the other four European

species are represented by 2–20 sequences. Second, the

De Lange et al. IMA Fungus (2021) 12:20 Page 27 of 31

UNITE nomenclature refers to, in our opinion, an incor-

rect concept of R.albonigra and there is a lack of means

with which we can assign potential correct and valid

available names to the North American taxa.

As the result of a concerted effort to improve UNITE

annotations, Nilsson et al. (2014) designated, based on

relevant literature data, 1368 species hypothesis reference

sequences but also marked 363 sequences of compro-

mised quality. Sequences of compromised technical

quality often originate from amplicon sequencing of envir-

onmental samples and are biased by the sequencing tech-

nique, and some of them can be recognised by automated

chimera search or presence of IUPAC ambiguity codes

(Badotti et al. 2017; Nilsson et al. 2018). Our comparison

of distinguishing nucleotide positions (Additional file 1)

demonstrated that short ITS sequences usually do not

provide sufficient information for species identification,

i.e. sequences UDB0502905 and UDB031025 containing

only ITS2 region are undoubtedly identified to species,

but UDB0663165 and UDB0557800 provide dubious in-

formation. The sequence UDB065518 does not match any

species recognised in our tree due to low quality or pos-

sibly it represents a new taxon. Our study dealt with five

species and we identified four sequences of compromised

quality, this is a higher ratio of low quality sequences

detected compared to the above mentioned study of

Nilsson et al. (2014).

Hofstetter et al. (2019) highlight the main problems with

sequence-based identification of fungi. Besides poor taxon

coverage in public sequence databases, misidentification,

the use of wrong names and bad annotation of sequences,

remain a major problem. As sequence-based identification

becomes more and more routine and the standard ap-

proach for many (mainly ecological) studies, it is clear that

the annotation of public sequences urgently needs to be

improved. An important implementation is the recent

introduction of taxonomic hypothesis that communicate

SH with taxonomic identification (Kõljalg et al. 2020). It

allows the tracing of taxonomic concepts presented at

UNITE and to link them with data about other fungal

traits. Hofstetter et al. (2019) and Durkin et al. (2020)state

that taxonomy finds itself at the same risk of extinction as

the very species they are supposed to study and provide

some recommendations towards fungal taxonomists on

how to highlight the importance and improve taxonomic

work. Taxonomists should create a reliable taxonomic

framework that can be used by conservationists and ecolo-

gists for sequence based identification. Lucking et al.

(2020) provides a discussion on how to improve the qual-

ity of identification tools and states that these tools are

only as good as the reference data behind them. The

UNITE database favours third-party annotation by taxo-

nomic experts to improve the taxonomic annotation of

the sequence data (Nilsson et al. 2018). Additional file 3

provides our proposed correction to the annotation of the

sequences, with the immediate advantage that it can be

directly integrated into further studies.

CONCLUSION

Russula albonigra has always been seen as one of the

more easily recognizable species within R. subgen. Com-

pactae by its strongly and rapidly blackening reaction of

the context, without intermediate reddening, resulting in a

strong black-and-white contrast of wounded and un-

touched parts, and the menthol-cooling taste of the lamel-

lae. The species was also thought to cover a broad

ecological amplitude and large distribution area. However,

molecular analysis revealed that R.albonigra s. lat.repre-

sents a species complex consisting of at least five Euro-

pean, two North American, and one Chinese species. A

thorough morphological study shows that the characters

traditionally used are not always reliable to define the R.

albonigra complex, instead the lack of a reaction of the

cystidial content to sulfovanillin is proposed as a character

to improve delimitation of the R. albonigra complex. This

feature is only shared with R. nigricans which can be read-

ily distinguished by the more spaced lamellae. Our UNITE

species hypothesis threshold testing revealed perfect

phylogenetic species match at a sequence distance thresh-

old of 0.5% for the R.albonigra complex. UNITE species

hypothesis may be a powerful tool to improve knowledge

about distribution and ecology of studied species, but the

pitfalls include short and low quality sequences. For

phylogenetic analyses of the ITS region we recommend

the use of sequences with existing (not blank) UNITE spe-

cies hypotheses at the 0.5% threshold and sequences of full

length. Our observations can be applicable for the genus

Russula as a whole and to many other macrofungal

genera. The importance of looking for the best threshold

value according to the fungal group of interest is

emphasised.

Supplementary Information

The online version contains supplementary material available at https://doi.

org/10.1186/s43008-021-00064-0.

Additional file 1. Sequence similarity table. Table showing sequence

similarity at distinguishing nucleotide positions.

Additional file 2. Comparison table. Table with a detailed comparison

of microscopical characters.

Additional file 3. Proposed corrected sequence annotation. Table with

proposed corrected annotation of the taxonomic data of the public

sequences.

Acknowledgements

Helga Marxmüller is thanked for providing material from her personal

herbarium. Jesko Kleine is thanked for providing material from his personal

herbarium and checking the taxonomy. Miroslav Caboňis thanked for his

suggestions to improve the phylogenetic analysis. Geoffrey Kibby is thanked

De Lange et al. IMA Fungus (2021) 12:20 Page 28 of 31

for proofreading the manuscript as a native English speaker. The Entoloma

team, organizing the week of fieldwork in Steinkjer, Norway in August 2016,

is acknowledged for allowing Annemieke Verbeken to participate and for

supporting her in the fieldwork.

Authors’contributions

RDL provided and gathered collections/sequences to constitute the final

dataset, performed part of the molecular lab work, performed the molecular

analysis, performed the microscopical study, made the descriptions for the

new species and the microscopic plates and wrote the manuscript. SA

provided collections and contributed to the macroscopical descriptions,

aided the microscopical study and molecular analysis, was involved in the

setup of the concept and the design of the study, was a major contributor

in writing the manuscript. KA and PA performed part of the molecular lab

work, KA annotated and submitted all sequences to the GenBank database.

JB provided collections/sequences essential to the study (the collection used

to determine the clade representing R.albonigra) and contributed to the

macroscopical descriptions. LD provided collections and performed part of

the molecular lab work. FH provided collections, contributed to the

macroscopical descriptions and performed part of the molecular lab work.

AV is the promotor of the first author, provided collections and contributed

to the macroscopical descriptions, was involved in the setup of the concept

and the design of the study, was a major contributor in writing the

manuscript. All authors reviewed the manuscript. All authors read and

approved the final manuscript.

Funding

LD (grant BOFDOC2015007001) was funded by a scholarship of the Special

Research Fund and by the JEC (Journées européennes du Cortinaire) 2016

organisation during the collecting of specimens. The lab work of SA and KA

is funded by the Slovak national project APVV 15–0210. The overall work of

JB was supported by the Long-term Development Projects RVO67985831

and RVO61389005.

Availability of data and materials

All data generated or analysed during this study are included in this

published article [and its supplementary information files].

Declarations

Adherence to national and international regulations

We confirm adherence to any pertinent national or international legislation

or regulations that apply to the transfer of living biotic materials used in the

study between countries.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Author details

Research Group Mycology, Department of Biology, Ghent University, K.L.

Ledeganckstraat 35, 9000 Ghent, Belgium.

Institute of Botany, Plant Science

and Biodiversity Center, Slovak Academy of Sciences, Dúbravská cesta 9, 845

23 Bratislava, Slovakia.

Institute of Forest Ecology Slovak Academy of

Sciences, Akademická 2, 949 01 Nitra, Slovakia.

Institute of Geology of the

Czech Academy of Sciences, Rozvojová 269, 165 00 Prague 6, Czech

Republic.

Nuclear Physics Institute of the Czech Academy of Sciences, Hlavní

130, 250 68 Husinec-Řež, Czech Republic.

Meise Botanic Garden, Research

Department, Nieuwelaan 38, 1860 Meise, Belgium.

Received: 26 November 2020 Accepted: 12 April 2021

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Full-text available

Sep 2020

Taxonomic mycology struggles with what seems to be a perpetual shortage of resources. Logically, fungal taxonomists should therefore leverage every opportunity to highlight and visualize the importance of taxonomic work, the usefulness of taxonomic data far beyond taxonomy, and the integrative and collabo-rative nature of modern taxonomy at large. Is mycology really doing that, though? In this study, we went through ten years' worth (2009-2018) of species descriptions of extant fungal taxa-1,097 studies describing at most ten new species-in five major mycological journals plus one plant journal. We estimated the frequency at which a range of key words, illustrations, and concepts related to ecology, geography, taxonomy, molecular data, and data availability were provided with the descriptions. We also considered a range of science-demographical aspects such as gender bias and the rejuvenation of taxonomy and tax-onomists as well as public availability of the results. Our results show that the target audience of fungal species descriptions appears to be other fungal taxonomists, because many aspects of the new species were presented only implicitly, if at all. Although many of the parameters we estimated show a gradual, and in some cases marked, change for the better over time, they still paint a somewhat bleak picture of mycologi-cal taxonomy as a male-dominated field where the wants and needs of an extended target audience are often not understood or even considered. This study hopes to leave a mark on the way fungal species are described by putting the focus on ways in which fungal taxonomy can better anticipate the end users of species descriptions-be they mycologists, other researchers, the public at large, or even algorithms. In the end, fungal taxonomy, too, is likely to benefit from such measures.

Convolutional neural networks improve fungal classification

Article

Full-text available

Jul 2020

Sequence classification plays an important role in metagenomics studies. We assess the deep neural network approach for fungal sequence classification as it has emerged as a successful paradigm for big data classification and clustering. Two deep learning-based classifiers, a convolutional neural network (CNN) and a deep belief network (DBN) were trained using our recently released barcode datasets. Experimental results show that CNN outperformed the traditional BLAST classification and the most accurate machine learning based Ribosomal Database Project (RDP) classifier on datasets that had many of the labels present in the training datasets. When classifying an independent dataset namely the “Top 50 Most Wanted Fungi”, CNN and DBN assigned less sequences than BLAST. However, they could assign much more sequences than the RDP classifier. In terms of efficiency, it took the machine learning classifiers up to two seconds to classify a test dataset while it was 53 s for BLAST. The result of the current study will enable us to speed up the taxonomic assignments for the fungal barcode sequences generated at our institute as ~ 70% of them still need to be validated for public release. In addition, it will help to quickly provide a taxonomic profile for metagenomics samples.

Unambiguous identification of fungi: Where do we stand and how accurate and precise is fungal DNA barcoding?

Article

Full-text available

Jul 2020

True fungi (Fungi) and fungus-like organisms (e.g. Mycetozoa, Oomycota) constitute the second largest group of organisms based on global richness estimates, with around 3 million predicted species. Compared to plants and animals, fungi have simple body plans with often morphologically and ecologically obscure structures. This poses challenges for accurate and precise identifications. Here we provide a conceptual framework for the identification of fungi, encouraging the approach of integrative (polyphasic) taxonomy for species delimitation, i.e. the combination of genealogy (phylogeny), phenotype (including autecology), and reproductive biology (when feasible). This allows objective evaluation of diagnostic characters, either phenotypic or molecular or both. Verification of identifications is crucial but often neglected. Because of clade-specific evolutionary histories, there is currently no single tool for the identification of fungi, although DNA barcoding using the internal transcribed spacer (ITS) remains a first diagnosis, particularly in metabarcoding studies. Secondary DNA barcodes are increasingly implemented for groups where ITS does not provide sufficient precision. Issues of pairwise sequence similarity-based identifications and OTU clustering are discussed, and multiple sequence alignment-based phylogenetic approaches with subsequent verification are recommended as more accurate alternatives. In metabarcoding approaches, the trade-off between speed and accuracy and precision of molecular identifications must be carefully considered. Intragenomic variation of the ITS and other barcoding markers should be properly documented, as phylotype diversity is not necessarily a proxy of species richness. Important strategies to improve molecular identification of fungi are: (1) broadly document intraspecific and intragenomic variation of barcoding markers; (2) substantially expand sequence repositories, focusing on undersampled clades and missing taxa; (3) improve curation of sequence labels in primary repositories and substantially increase the number of sequences based on verified material; (4) link sequence data to digital information of voucher specimens including imagery. In parallel, technological improvements to genome sequencing offer promising alternatives to DNA barcoding in the future. Despite the prevalence of DNA-based fungal taxonomy, phenotype-based approaches remain an important strategy to catalog the global diversity of fungi and establish initial species hypotheses.

MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets

Article

Full-text available

Jul 2016

We present the latest version of the Molecular Evolutionary Genetics Analysis (MEGA) software, which contains many sophisticated methods and tools for phylogenomics and phylomedicine. In this major upgrade, MEGA has been optimized for use on 64-bit computing systems for analyzing bigger datasets. Researchers can now explore and analyze tens of thousands of sequences in MEGA. The new version also provides an advanced wizard for building timetrees and includes a new functionality to automatically predict gene duplication events in gene family trees. The 64-bit MEGA is made available in two interfaces: graphical and command line. The graphical user interface (GUI) is a native Microsoft Windows application that can also be used on Mac OSX. The command line MEGA is available as native applications for Windows, Linux, and Mac OSX. They are intended for use in high-throughput and scripted analysis. Both versions are available from www.megasoftware.net free of charge.

One step closer to unravelling the origin of Russula: subgenus Glutinosae subg. nov

Article

Full-text available

Feb 2020

This study reports on the discovery of a new subgenus, Russula subg. Glutinosae, having an Eastern North American-East Asian distribution. A multigene phylogeny places this new subgenus sister with strong support to a well-supported clade composed of subgenera Compactae and Archaeae. It holds only two very rare, northern hemisphere species, the North American R. glutinosa and the Asian R. glutinosoides sp. nov., thereby adding support to a northern hemisphere origin of the genus. Russula fattoensis is here shown to be a synonym of R. glutinosa. Detailed morphological descriptions and illustrations of holotype collections are provided and potential affinities and similarities with other subgenera are discussed. The new subgenus is a perfect illustration of the fact that nBLAST of nrITS does not always provide the appropriate sampling for phylogenetic analyses.

Fungal evolution: diversity, taxonomy and phylogeny of the Fungi

Article

Full-text available

Dec 2019

The fungal kingdom comprises a hyperdiverse clade of heterotrophic eukaryotes characterized by the presence of a chitinous cell wall, the loss of phagotrophic capabilities and cell organizations that range from completely unicellular monopolar organisms to highly complex syncitial filaments that may form macroscopic structures. Fungi emerged as a ‘Third Kingdom’, embracing organisms that were outside the classical dichotomy of animals versus vegetals. The taxonomy of this group has a turbulent history that is only now starting to be settled with the advent of genomics and phylogenomics. We here review the current status of the phylogeny and taxonomy of fungi, providing an overview of the main defined groups. Based on current knowledge, nine phylum‐level clades can be defined: Opisthosporidia, Chytridiomycota, Neocallimastigomycota, Blastocladiomycota, Zoopagomycota, Mucoromycota, Glomeromycota, Basidiomycota and Ascomycota. For each group, we discuss their main traits and their diversity, focusing on the evolutionary relationships among the main fungal clades. We also explore the diversity and phylogeny of several groups of uncertain affinities and the main phylogenetic and taxonomical controversies and hypotheses in the field.

The Quest for a Globally Comprehensible Russula Language

Article

Full-text available

Oct 2019
FUNGAL DIVERS

Since 2007, the quality of Russula descriptions has improved and the use of molecular support for species delimitation and the number of published new species has increased. However, the description style is not consistent and has regional or author-specific patterns. Most recent publications still favour descriptions of spores compared to hymenium and pileipellis elements, and usually only the spore size is provided with statistical support. This study proposes standards for descriptions of the microscopic structure of Russula species (Russulaceae, Agaricomycetes). We present the description template, the template measurements table, the specific terminology and the essential chemical reagents. The proposed standards were tested by mycologists from 11 countries who met at the Russula Microscopy Workshop in Slovakia. Descriptions of 26 species from 9 countries and four continents were prepared, among them R. amarissima, R. castanopsidis, R. seperina and R. subtilis are re-described and 15 species are introduced as new: R. abietiphila, R. amerorecondita, R. aurantioflava, R. echidna, R. flavobrunnescens, R. fluvialis, R. fortunae, R. garyensis, R. gemmata, R. laevis, R. madrensis, R. olivaceohimalayensis, R. purpureogracilis, R. sancti-pauli and R. wielangtae. Seven descriptions for candidate new species are provided without a formal name assignment. Pairwise comparison of species described in this study with available similar descriptions of related species suggests that microscopic characters from all parts of the basidiomata can be equally important for species recognition and they deserve the same treatment including number of measurements and statistics. The majority of recent studies does not recognise differences between the pileus margin and centre, but more than one-third of the species described in this study show distinct differences between the pileus areas, emphasizing the importance to specify the origin of pileipellis observations. This study proved that there is frequently insufficient difference in the ITS barcode between closely related species and that it is necessary to use more genetic markers combined with ecological and geographical data.

Looks can be deceiving: the deceptive milkcaps (Lactifluus, Russulaceae) exhibit low morphological variance but harbour high genetic diversity

Article

Full-text available

Dec 2019

Abstract The ectomycorrhizal genus Lactifluus is known to contain many species complexes, consisting of morphologically very similar species, which can be considered cryptic or pseudocryptic. In this paper, a thorough molecular study is performed of the clade around Lactifluus deceptivus (originally described by Peck from North America) or the deceptive milkcaps. Even though most collections were identified as L. deceptivus, the clade is shown to contain at least 15 species, distributed across Asia and America, indicating that the L. deceptivus clade represents a species complex. These species are morphologically very similar and are characterized by a tomentose pileus with thin-walled hyphae and a velvety stipe with thick-walled hyphae. An ITS1 sequence was obtained through Illumina sequencing for the lectotype of L. deceptivus, dating from 1885, revealing which clade represents the true L. deceptivus. In addition, it is shown that three other described species also belong to the L. deceptivus clade: L. arcuatus, L. caeruleitinctus and L. mordax, and molecularly confirmed that L. tomentoso-marginatus represents a synonym of L. deceptivus. Furthermore, two new Neotropical species are described: Lactifluus hallingii and L. domingensis.

Coalescent-based delimitation and species-tree estimations reveal Appalachian origin and Neogene diversification in Russula subsection Roseinae

Article

Mar 2020

Numerous lineages of mushroom-forming fungi have been subject to bursts of diversification throughout their evolutionary history, events that can impact our ability to infer well-resolved phylogenies. However, groups that have undergone quick genetic change may have the highest adaptive potential. As the second largest genus of mushroom-forming fungi, Russula provides an excellent model for studying hyper-diversification and processes in evolution that drives it. This study focuses on the morphologically defined group – Russula subsection Roseinae. Species hypotheses based on morphological differentiation and multi-locus phylogenetic analyses are tested in the Roseinae using different applications of the multi-species coalescent model. Based on this combined approach, we recognize fourteen species in Roseinae including the Albida and wholly novel Magnarosea clades. Reconstruction of biogeographic and host association history suggest that parapatric speciation in refugia during glacial cycles of the Pleistocene drove diversification within the Roseinae, which is found to have a Laurasian distribution with an evolutionary origin in the Appalachian Mountains of eastern North America. Finally, we detect jump dispersal at a continental scale that has driven diversification since the most recent glacial cycles.

Enlightening the black and white: species delimitation and UNITE species hypothesis testing in the Russula albonigra species complex

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